Immunomodulatory therapeutic mrna compositions encoding activating oncogene mutation peptides

ABSTRACT

The disclosure features immunomodulatory therapeutic compositions of an mRNA encoding an activating oncogene mutation peptide and an mRNA encoding a polypeptide that enhances immune responses to the activating oncogene mutation peptide, for example an mRNA encoding an immune potentiator. The disclosure also features methods of using the same, for example, to stimulate anti-cancer immune responses.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 62/453,465, filed on Feb. 1, 2017; U.S. Provisional Application Ser.No. 62/467,063, filed on Mar. 3, 2017; U.S. Provisional Application Ser.No. 62/490,523, filed on Apr. 26, 2017; and U.S. Provisional ApplicationSer. No. 62/541,571, filed on Aug. 4, 2017. The entire contents of theabove-referenced applications are incorporated herein by this reference.

BACKGROUND OF THE DISCLOSURE

The ability to modulate an immune response is beneficial in a variety ofclinical situations, including the treatment of cancer and pathogenicinfections, as well as in potentiating vaccine responses to provideprotective immunity. A number of therapeutic tools exist for modulatingthe function of biological pathways and/or molecules that are involvedin diseases such as cancer and pathogenic infections. These toolsinclude, for example, small molecule inhibitors, cytokines andtherapeutic antibodies. Some of these tools function through modulatingimmune responses in a subject, such as cytokines that modulate theactivity of cells within the immune system or immune checkpointinhibitor antibodies, such as anti-CTLA-4 or anti-PD-L1 that modulatethe regulation of immune responses.

Additionally, vaccines have long been used to stimulate an immuneresponse against antigens of pathogens to thereby provide protectiveimmunity against later exposure to the pathogens. More recently,vaccines have been developed using antigens found on tumor cells tothereby enhance anti-tumor imunoresponsiveness. In addition to theantigen(s) used in the vaccine, other agents may be included in avaccine preparation, or used in combination with the vaccinepreparation, to further boost the immune response to the vaccine. Suchagents that enhance vaccine responsiveness are referred to in the art asadjuvants. Examples of commonly used vaccine adjuvants include aluminumgels and salts, monophosphoryl lipid A, MF59 oil-in-water emulsion,Freund's complete adjuvant, Freund's incomplete adjuvant, detergents andplant saponins. These adjuvants typically are used with protein orpeptide based vaccines. Alternative types of vaccines, such as RNA basedvaccines, are now being developed.

There exists a need in the art for additional effective agents thatenhance immune responses to an antigen of interest.

SUMMARY OF THE DISCLOSURE

Provided herein are immunomodulatory therapeutic compositions, includinglipid-based compositions such as lipid nanoparticles, which include anRNA (e.g., messenger RNA (mRNA)) that can safely direct the body'scellular machinery to produce a cancer protein or fragment thereof ofinterest, e.g., an activating oncogene mutation peptide. In someembodiments, the RNA is a modified RNA. The immunomodulatory therapeuticcompositions, including mRNA compositions and/or lipid nanoparticlescomprising the same are useful to induce a balanced immune responseagainst cancers, comprising both cellular and humoral immunity, withoutrisking the possibility of insertional mutagenesis, for example.

The immunomodulatory therapeutic compositions, including mRNAcompositions and/or lipid nanoparticles of the disclosure may beutilized in various settings depending on the prevalence of the canceror the degree or level of unmet medical need. The immunomodulatorytherapeutic compositions, including mRNA compositions and lipidnanoparticles of the disclosure may be utilized to treat and/or preventa cancer of various stages or degrees of metastasis. Theimmunomodulatory therapeutic compositions and lipid nanoparticles of thedisclosure have superior properties in that they produce much largerantibody titers and produce responses earlier than alternativeanti-cancer therapies including cancer vaccines. While not wishing to bebound by theory, it is believed that the provided compositions, such asmRNA polynucleotides, are better designed to produce the appropriateprotein conformation upon translation as the RNA co-opt natural cellularmachinery. Unlike traditional therapies and vaccines which aremanufactured ex vivo and may trigger unwanted cellular responses, RNA ofthe provided compositions are presented to the cellular system in a morenative fashion.

In some aspects, the disclosure provides an immunomodulatory therapeuticcomposition, comprising: one or more mRNA each comprising an openreading frame encoding an activating oncogene mutation peptide, andoptionally one or more mRNA each comprising an open reading frameencoding a polypeptide that enhances an immune response to theactivating oncogene mutation peptide in a subject, wherein the immuneresponse comprises a cellular or humoral immune response characterizedby: (i) stimulating Type I interferon pathway signaling, (ii)stimulating NFkB pathway signaling, (iii) stimulating an inflammatoryresponse, (iv) stimulating cytokine production, (v) stimulatingdendritic cell development, activity or mobilization, and (vi) acombination of any of (i)-(v); and a pharmaceutically acceptablecarrier.

In some aspects, the disclosure provides an immunomodulatory therapeuticcomposition, including mRNA compositions and/or lipid nanoparticlescomprising the same, that enhances an immune response by, for example,stimulating Type I interferon pathway signaling, stimulating NFkBpathway signaling, stimulating an inflammatory response, stimulatingcytokine production or stimulating dendritic cell development, activityor mobilization. Enhancement of an immune response to an antigen ofinterest by an immune potentiator mRNA results in, for example,stimulation of cytokine production, stimulation of cellular immunity (Tcell responses), such as antigen-specific CD8⁺ or CD4⁺ T cell responsesand/or stimulation of humoral immunity (B cell responses), such asantigen-specific antibody responses.

In some aspects, the disclosure provides an immunomodulatory therapeuticcomposition wherein the activating oncogene mutation is a KRAS mutation.In some aspects, the KRAS mutation is a G12 mutation. In some aspects,the G12 KRAS mutation is selected from G12D, G12V, G12S, G12C, G12A, andG12R KRAS mutations. In other aspects, the G12 KRAS mutation is selectedfrom G12D, G12V, and G12C KRAS mutations. In some aspects, the KRASmutation is a G13 mutation. In some aspects, the G13 KRAS mutation is aG13D KRAS mutation. In other aspects, the disclosure provides animmunomodulatory therapeutic composition wherein the activating oncogenemutation is a H-RAS or N-RAS mutation.

In some embodiments the skilled artisan will select a KRAS mutation, aHLA subtype and a tumor type based on the guidance provided herein andprepare a KRAS vaccine for therapy. In some embodiments the KRASmutation is selected from: G12C, G12V, G12D, G13D. In some embodimentsthe HLA subtype is selected from: A*02:01, C*07:01, C*04:01, C*07:02,HLA-A11 and/or HLA-C08. In some embodiments the tumor type is selectedfrom colorectal, pancreatic, lung (e.g., non-small cell lung cancer(NSCLC), and endometrioid.

In some embodiments, the HRAS mutation is a mutation at codon 12, codon13, or codon 61. In some embodiments, the HRAS mutation is a 12V, 61L,or 61R mutation.

In some embodiments, the NRAS mutation is a mutation at codon 12, codon13, or codon 61. In some embodiments, the NRAS mutation is a 12D, 13D,61K, or 61R mutation.

In some aspects, the disclosure provides an immunomodulatory therapeuticcomposition of any one of the foregoing or related embodiments, whereinthe mRNA has an open reading frame encoding a concatemer of two or moreactivating oncogene mutation peptides. In some aspects, the concatemercomprises 3, 4, 5, 6, 7, 8, 9, or 10 activating oncogene mutationpeptides. In some aspects, the concatemer comprises 4 activatingoncogene mutation peptides.

In other aspects, the disclosure provides an immunomodulatorytherapeutic composition, comprising: an mRNA comprising an open readingframe encoding a concatemer of two or more activating oncogene mutationpeptides, wherein the concatemer comprises KRAS activating oncogenemutation peptides G12D, G12V, G12C, and G13D; and one or more mRNA eachcomprising an open reading frame encoding a polypeptide that enhances animmune response to the KRAS activating oncogene mutation peptides in asubject. In some aspects, the concatemer comprises from N- to C-terminusG12D, G12V, G13D, and G12C. In some aspects, the concatemer comprisesfrom N- to C-terminus G12C, G13D, G12V, and G12D.

Some embodiments of the present disclosure provide immunomodulatorytherapeutic compositions that include an mRNA comprising an open readingframe encoding a concatemer of two or more activating oncogene mutationpeptides. In some embodiments, at least two of the peptide epitopes areseparated from one another by a single Glycine. In some embodiments, theconcatemer comprises 3-10 activating oncogene mutation peptides. In somesuch embodiments, all of the peptide epitopes are separated from oneanother by a single Glycine. In other embodiments, at least two of thepeptide epitopes are linked directly to one another without a linker.

In some aspects, the disclosure provides an immunomodulatory therapeuticcomposition, comprising: 1, 2, 3, or 4 mRNAs encoding 1, 2, 3, or 4activating oncogene mutation peptides; and one or more mRNA eachcomprising an open reading frame encoding a polypeptide that enhances animmune response to the activating oncogene mutation peptide in asubject. In some aspects, the composition comprises 4 mRNAs encoding 4activating oncogene mutation peptides. In some aspects, the 4 mRNAsencode KRAS activating oncogene mutation peptides G12D, G12V, G12C, andG13D.

In some aspects, the disclosure provides an immunomodulatory therapeuticcomposition of any one of the foregoing or related embodiments, whereinthe activating oncogene mutation peptide comprises 10-30, 15-25, or20-25 amino acids in length. In some aspects, the activating oncogenemutation peptide comprises 20, 21, 22, 23, 24, or 25 amino acids inlength. In some aspects, the activating oncogene mutation peptidecomprises 25 amino acids in length.

In some aspects, the disclosure provides an immunomodulatory therapeuticcomposition of any one of the foregoing or related embodiments, whereinthe mRNA encoding a polypeptide that enhances an immune response to theactivating oncogene mutation peptide in a subject encodes aconstitutively active human STING polypeptide. In some aspects, theconstitutively active human STING polypeptide comprises one or moremutations selected from the group consisting of V147L, N154S, V155M,R284M, R284K, R284T, E315Q, R375A, and combinations thereof.

In some aspects, the constitutively active human STING polypeptidecomprises mutation V155M (e.g., having the amino acid sequence shown inSEQ ID NO: 1 or encoded by a nucleotide sequence shown in SEQ ID NO: 139or 170). In some aspects the constitutively active human STINGpolypeptide comprises mutations V147L/N154S/V155M. In some aspects, theconstitutively active human STING polypeptide comprises mutationsR284M/V147L/N154S/V155M.

In other aspects, the constitutively active human STING polypeptidecomprises an amino acid sequence set forth in any one of SEQ ID NOs:1-10 and 164. In another aspect, the constitutively active human STINGpolypeptide is encoded by a nucleotide sequence set forth in any one ofSEQ ID NOs: 139-148, 165, 168, 170, 201-209 and 225. In some aspects,the constitutively active human STING polypeptide comprises a 3′ UTRcomprising at least one miR-122 microRNA binding site, such as forexample set forth in SEQ ID NO: 149.

In some aspects, the disclosure provides an immunomodulatory therapeuticcomposition of any one of the foregoing or related embodiments, whereinthe mRNA encoding a polypeptide that enhances an immune response to theactivating oncogene mutation peptide in a subject encodes aconstitutitively active human IRF3 polypeptide. In one aspect, theconstitutively active human IRF3 polypeptide comprises an S396Dmutation. In one aspect, the constitutively active human IRF3polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 12or is encoded by a nucleotide sequence set forth in SEQ ID NO: 151 or212. In one aspect, the constitutively active IRF3 polypeptide is amouse IRF3 polypeptide, for example comprising an amino acid sequenceset forth in SEQ ID NO: 11 or encoded by the nucleotide sequence shownin SEQ ID NO: 150 or 211.

In some aspects, the disclosure provides an immunomodulatory therapeuticcomposition of any one of the foregoing or related embodiments, whereinthe mRNA encoding a polypeptide that enhances an immune response to theactivating oncogene mutation peptide in a subject encodes aconstitutitively active human IRF7 polypeptide. In one aspect, theconstitutively active human IRF7 polypeptide comprises one or moremutations selected from the group consisting of S475D, S476D, S477D,S479D, L480D, S483D, S487D, and combinations thereof; deletion of aminoacids 247-467; and combinations of the foregoing mutations and/ordeletions. In one embodiment, the constitutively active human IRF7polypeptide comprises an amino acid sequence set forth in any one of SEQID NOs: 14-18. In one embodiment, the constitutively active human IRF7polypeptide is encoded by a nucleotide sequence set forth in any one ofSEQ ID NOs: 153-157 and 214-218.

In yet other aspects, the disclosure provides an immune potentiator mRNAencoding a polypeptide selected from the group consisting of MyD88,TRAM, IRF1, IRF8, IRF9, TBK1, IKKi, STAT1, STAT2, STAT4, STAT6, c-FLIP,IKKβ, RIPK1, TAK-TAB1 fusion, DIABLO, Btk, self-activating caspase-1 andFlt3.

In some aspects, the disclosure provides an immunomodulatory therapeuticcomposition of any one of the foregoing embodiments, wherein thecomposition further comprises a cancer therapeutic agent. In someaspects, the composition further comprises an inhibitory checkpointpolypeptide. In some aspects, the inhibitory checkpoint polypeptide isan antibody or fragment thereof that specifically binds to a moleculeselected from the group consisting of PD-1, PD-L1, TIM-3, VISTA, A2AR,B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR and LAG3.

In other embodiments, the composition further comprises a recallantigen. For example, in some embodiments, the recall antigen is aninfectious disease antigen.

In some embodiments, the composition does not comprise a stabilizationagent.

In some aspects, the disclosure provides an immunomodulatory therapeuticcomposition of any one of the foregoing embodiments, wherein the mRNA isformulated in a lipid nanoparticle. In some aspects, the lipidnanoparticle comprises a molar ratio of about 20-60% ionizable aminolipid:5-25% phospholipid:25-55% sterol; and 0.5-15% PEG-modified lipid.In some aspects, the inonizable amino lipid is selected from the groupconsisting of for example,2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319). In someaspects, the ionizable amino lipid comprises a compound of any ofFormulae (I), (IA), (II), (IIa), (IIb), (IIc), (IId), and (IIe). In someaspects, the ionizable amino lipid comprises a compound of Formula (I).In some aspects, the compound of Formula (I) is Compound 25.

In some aspects, the disclosure provides an immunomodulatory therapeuticcomposition of any one of the foregoing embodiments, wherein each mRNAincludes at least one chemical modification. In some aspects, thechemical modification is selected from the group consisting ofpseudouridine, N1-methylpseudouridine, 2-thiouridine, 4′-thiouridine,5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine,2-thio-dihydropseudouridine, 2-thio-dihydrouridine,2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine,4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine,4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine,5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2′-O-methyluridine. In some aspects, the chemical modification is pseudouridine ora pseudouridine analog. In some aspects, the chemical modification isN1-methylpseudouridine. In some aspects, each mRNA comprises fullymodified N1-methylpseudouridine.

In some aspects, the disclosure provides an immunomodulatory therapeuticcomposition, including mRNA compositions and lipid-based compositionssuch as lipid nanoparticles, comprising: one or more mRNA eachcomprising an open reading frame encoding a KRAS activating oncogenemutation peptide, and optionally one or more mRNA each comprising anopen reading frame encoding a constitutively active human STINGpolypeptide; and a pharmaceutically acceptable carrier. In some aspects,the constitutively active human STING polypeptide comprises mutationV155M. In some aspects, the constitutively active human STINGpolypeptide comprises an amino acid sequence shown in SEQ ID NO: 1. Insome aspects, the constitutively active human STING polypeptidecomprises a 3′ UTR comprising at least one miR-122 microRNA bindingsite.

In some aspects, the disclosure provides an immunomodulatory therapeuticcomposition of any one of the foregoing embodiments, wherein the KRASactivating oncogene mutation peptide is selected from G12D, G12V, G12S,G12C, G12A, G12R, and G13D. In some aspects, the KRAS activatingoncogene mutation peptide is selected from G12D, G12V, G12C, and G13D.

In some aspects, the disclosure provides an immunomodulatory therapeuticcomposition of any one of the foregoing embodiments, wherein the mRNAcomprises an open reading frame encoding a concatemer of two or moreKRAS activating oncogene mutation peptides. In some aspects, theconcatemer comprises 3, 4, 5, 6, 7, 8, 9 or 10 KRAS activating oncogenemutation peptides. In some aspects, the concatemer comprises 4 KRASactivating oncogene mutation peptides. In some aspects, the concatemercomprises G12D, G12V, G12C, and G13D. In some aspects, the concatemercomprises from N- to C-terminus G12D, G12V, G13D, and G12C. In someaspects, the concatemer comprises from N- to C-terminus G12C, G13D,G12V, and G12D.

In some aspects, the disclosure provides an immunomodulatory therapeuticcomposition of any one of the foregoing embodiments, wherein thecomposition comprises 1, 2, 3, or 4 mRNAs encoding 1, 2, 3, or 4 KRASactivating oncogene mutation peptides. In some aspects, the compositioncomprises 4 mRNAs encoding 4 KRAS activating oncogene mutation peptides.In some aspects, the 4 KRAS activating oncogene mutation peptidescomprise G12D, G12V, G12C, and G13D.

In some aspects, the disclosure provides an immunomodulatory therapeuticcomposition of any one of the foregoing or related embodiments, whereinthe KRAS activating oncogene mutation peptide comprises 10-30, 15-25, or20-25 amino acids in length. In some aspects, the KRAS activatingoncogene mutation peptide comprises 20, 21, 22, 23, 24, or 25 aminoacids in length. In some aspects, the activating oncogene mutationpeptide comprises 25 amino acids in length.

In some aspects, the disclosure provides an immunomodulatory therapeuticcomposition of any one of the foregoing or related embodiments, whereinthe mRNA has an open reading frame encoding a concatemer of two or moreKRAS activating oncogene mutation peptides and the concatemer comprisesan amino acid sequence selected from the group set forth in SEQ ID NOS:42-47, 73 and 137. In some aspects, wherein the mRNA encoding theconcatemer comprises a nucleotide sequence selected from the group setforth in SEQ ID NOS: 129-131, 133, 138, 167, 169, 193-195 and 197-198.

In some aspects, the disclosure provides an immunomodulatory therapeuticcomposition of any one of the foregoing or related embodiments, whereinthe composition comprises 1, 2, 3, or 4 mRNAs encoding 1, 2, 3, or 4KRAS activating oncogene mutation peptides, and wherein the KRASactivating oncogene mutation peptides comprise an amino acid sequenceselected from the group set forth in SEQ ID NOs: 36-41, 72 and 125. Insome aspects, the KRAS activating oncogene mutation peptides comprisethe amino acid sequence set forth in SEQ ID NOs: 39-41 and 72. In someaspects, the mRNA encoding the KRAS activating oncogene mutation peptidecomprises a nucleotide sequence selected from the group set forth in SEQID NOs: 126-128, 132, 190-192 and 196.

In other aspects, the disclosure provides an immunomodulatorytherapeutic composition, including mRNA compositions and/or lipidnanoparticles comprising the same, comprising an mRNA construct encodingat least one mutant human KRAS antigen and a constitutively active humanSTING polypeptide, for example wherein the mRNA (e.g., a modified mRNA)encodes an amino acid sequence as set forth in any one of SEQ ID NOs:48-71.

In some aspects, the disclosure provides an immunomodulatory therapeuticcomposition of any one of the foregoing or related embodiments, whereineach mRNA is formulated in the same or different lipid nanoparticle. Insome aspects, each mRNA encoding a KRAS activating oncogene mutationpeptide is formulated in the same or different lipid nanoparticle. Insome aspects, each mRNA encoding constitutively active human STING isformulated in the same or different lipid nanoparticle. In some aspects,each mRNA encoding a KRAS activating oncogene mutation peptide isformulated in the same lipid nanoparticle and each mRNA encodingconstitutively active human STING is formulated in a different lipidnanoparticle. In some aspects, each mRNA encoding a KRAS activatingoncogene mutation peptide is formulated in the same lipid nanoparticleand each mRNA encoding constitutively active human STING is formulatedin the same lipid nanoparticle as each mRNA encoding a KRAS activatingoncogene mutation peptide. In some aspects, each mRNA encoding a KRASactivating oncogene mutation peptide is formulated in a different lipidnanoparticle and each mRNA encoding constitutively active human STING isformulated in the same lipid nanoparticle as each mRNA encoding eachKRAS activating oncogene mutation peptide.

In some aspects, the disclosure provides an immunomodulatory therapeuticcomposition of any one of the foregoing embodiments, wherein theimmunomodulatory therapeutic composition is formulated in a lipidnanoparticle, wherein the lipid nanoparticle comprises a molar ratio ofabout 20-60% ionizable amino lipid:5-25% phospholipid:25-55% sterol; and0.5-15% PEG-modified lipid. In some aspects, the ionizable amino lipidis selected from the group consisting of for example,2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319). In someaspects, the ionizable amino lipid comprises a compound of any ofFormulae (I), (IA), (II), (IIa), (IIb), (IIc), (IId), and (IIe). In someaspects, the ionizable amino lipid comprises a compound of Formula (I).In some aspects, the compound of Formula (I) is Compound 25.

In certain embodiments, the lipid nanoparticle comprises Compound 25 (asthe ionizable amino lipid), DSPC (as the phospholipid), cholesterol (asthe sterol) and PEG-DMG (as the PEG-modified lipid). In certainembodiments, the lipid nanoparticle comprises a molar ratio of about20-60% Compound 25:5-25% DSPC:25-55% cholesterol; and 0.5-15% PEG-DMG.In one embodiment, the lipid nanoparticle comprises a molar ratio ofabout 50% Compound 25:about 10% DSPC:about 38.5% cholesterol:about 1.5%PEG-DMG (i.e., Compound 25:DSPC:cholesterol:PEG-DMG at about a50:10:38.5:1.5 ratio). In one embodiment, the lipid nanoparticlecomprises a molar ratio of 50% Compound 25:10% DSPC:38.5% cholesterol:1.5% PEG-DMG (i.e., Compound 25:DSPC:cholesterol:PEG-DMG at a50:10:38.5:1.5 ratio).

In some aspects, the disclosure provides an immunomodulatory therapeuticcomposition of any one of the foregoing or related embodiments, whereineach mRNA includes at least one chemical modification. In some aspects,the chemical modification is selected from the group consisting ofpseudouridine, N1-methylpseudouridine, 2-thiouridine, 4′-thiouridine,5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine,2-thio-dihydropseudouridine, 2-thio-dihydrouridine,2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine,4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine,4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine,5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2′-O-methyluridine. In some aspects, the chemical modification is pseudouridine ora pseudouridine analog. In some aspects, the chemical modification isN1-methylpseudouridine. In some aspects, each mRNA comprises fullymodified N1-methylpseudouridine.

In some aspects, the disclosure provides a lipid nanoparticlecomprising: an mRNA comprising an open reading frame encoding aconcatemer of 4 KRAS activating oncogene mutation peptides, wherein the4 KRAS activating oncogene mutation peptides comprise G12D, G12V, G12C,and G13D; an mRNA comprising an open reading frame encoding aconstitutively active human STING polypeptide. In some aspects, theconcatemer comprises from N- to C-terminus G12D, G12V, G13D, and G12C.In some aspects, the concatemer comprises from N- to C-terminus G12C,G13D, G12V, and G12D.

In some aspects, the disclosure provides lipid nanoparticle of any oneof the foregoing embodiments, wherein each KRAS activating oncogenemutation peptide comprises 20, 21, 22, 23, 24, or 25 amino acids inlength. In some aspects, each KRAS activating oncogene mutation peptidecomprises 25 amino acids in length.

In some aspects, the disclosure provides a lipid nanoparticlecomprising: an mRNA comprising an open reading frame encoding aconcatemer of 4 KRAS activating oncogene mutation peptides, wherein the4 KRAS activating oncogene mutation peptides comprise G12D, G12V, G12C,and G13D, and wherein the concatemer comprises the amino acid sequenceset forth in SEQ ID NO:137; an mRNA comprising an open reading frameencoding a constitutively active human STING polypeptide. In someaspects, the mRNA encoding the concatemer of 4 KRAS activating oncogenemutation peptides comprises the nucleotide sequence set forth in SEQ IDNO: 138, SEQ ID NO: 167 or SEQ ID NO: 169. In some aspects, theconstitutively active human STING polypeptide comprises mutation V155M.In some aspects, the constitutively active human STING polypeptidecomprises the amino acid sequence shown in SEQ ID NO: 1. In someaspects, the mRNA encoding the constitutively active human STINGpolypeptide comprises a 3′ UTR comprising at least one miR-122 microRNAbinding site. In some aspects, the mRNA encoding the constitutivelyactive human STING polypeptide comprises the nucleotide sequence shownin SEQ ID NO: 139, SEQ ID NO: 168, or SEQ ID NO: 170.

In other aspects, the disclosure provides a lipid nanoparticlecomprising:

a first mRNAs comprising an open reading frame encoding a KRASactivating oncogene mutation peptide comprising G12D;

a second mRNA comprising an open reading frame encoding a KRASactivating oncogene mutation peptide comprising G12V;

a third mRNA comprising an open reading frame encoding a KRAS activatingoncogene mutation peptide comprising G12C;

a fourth mRNA comprising an open reading frame encoding a KRASactivating oncogene mutation peptide comprising G13D; and

a fifth mRNA comprising an open reading frame encoding a constitutivelyactive human STING polypeptide. In certain embodiments, the mRNAs arepresent at a KRAS:STING mass ratio selected from the group consisting of1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 and 10:1. In one embodiment,the mRNAs are present at a KRAS:STING mass ratio of 5:1.

In some aspects of the foregoing lipid nanoparticle, each KRASactivating oncogene mutation peptide comprises 20, 21, 22, 23, 24, or 25amino acids in length. In some aspects, each KRAS activating oncogenemutation peptide comprises 25 amino acids in length.

In some aspects of the foregoing lipid nanoparticle, the KRAS activatingoncogene mutation peptides comprise the amino acid sequences set forthin SEQ ID NOs: 39-41 and 72. In some aspects, the mRNAs encoding theKRAS activating oncogene mutation peptides comprise the nucleotidesequences set forth in SEQ ID NOs: 126-128, 132, 190-192 and 196.

In some aspects of the foregoing lipid nanoparticle, the constitutivelyactive human STING polypeptide comprises mutation V155M. In someaspects, the constitutively active human STING polypeptide comprises theamino acid sequence shown in SEQ ID NO: 1. In some aspects, the mRNAencoding the constitutively active human STING polypeptide comprises a3′ UTR comprising at least one miR-122 microRNA binding site. In someaspects, the mRNA encoding the constitutively active human STINGpolypeptide comprises the nucleotide sequence shown in SEQ ID NO: 139,SEQ ID NO: 168, or SEQ ID NO: 170.

In some aspects, the disclosure provides a lipid nanoparticle of any oneof the foregoing embodiments, wherein the lipid nanoparticle comprises amolar ratio of about 20-60% ionizable amino lipid:5-25%phopholipid:25-55% sterol; and 0.5-15% PEG-modified lipid. In someaspects, the inonizable amino lipid is selected from the groupconsisting of for example,2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319). In someaspects, the ionizable amino lipid comprises a compound of any ofFormulae (I), (IA), (II), (IIa), (IIb), (IIc), (IId), and (IIe). In someaspects, the ionizable amino lipid comprises a compound of Formula (I).In some aspects, the compound of Formula (I) is Compound 25.

In some aspects, the disclosure provides a lipid nanoparticle of any oneof the foregoing embodiments, wherein each mRNA includes at least onechemical modification. In some aspects, the chemical modification isselected from the group consisting of pseudouridine,N1-methylpseudouridine, 2-thiouridine, 4′-thiouridine, 5-methylcytosine,2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine,2-thio-5-aza-uridine, 2-thio-dihydropseudouridine,2-thio-dihydrouridine, 2-thio-pseudouridine,4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine,4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine,dihydropseudouridine, 5-methyluridine, 5-methyluridine,5-methoxyuridine, and 2′-O-methyl uridine. In some aspects, the chemicalmodification is pseudouridine or a pseudouridine analog. In someaspects, the chemical modification is N1-methylpseudouridine. In someaspects, each mRNA comprises fully modified N1-methylpseudouridine.

In some aspects, the disclosure provides a drug product comprising anyof the foregoing or related lipid nanoparticles for use in cancertherapy, optionally with instructions for use in cancer therapy.

In other aspects, the disclosure provides a first lipid nanoparticlecomprising: an mRNA comprising an open reading frame encoding a KRASactivating oncogene mutation peptide comprising G12D; and an mRNAcomprising an open reading frame encoding a constitutively active humanSTING polypeptide.

In some aspects, the disclosure provides a second lipid nanoparticlecomprising: an mRNA comprising an open reading frame encoding a KRASactivating oncogene mutation peptide comprising G12V; and an mRNAcomprising an open reading frame encoding a constitutively active humanSTING polypeptide.

In some aspects, the disclosure provides a third lipid nanoparticlecomprising an mRNA comprising an open reading frame encoding a KRASactivating oncogene mutation peptide comprising G12C; and an mRNAcomprising an open reading frame encoding a constitutively active humanSTING polypeptide.

In some aspects, the disclosure provides a fourth lipid nanoparticlecomprising: an mRNA comprising an open reading frame encoding a KRASactivating oncogene mutation peptide comprising G13D; and an mRNAcomprising an open reading frame encoding a constitutively active humanSTING polypeptide.

In some aspects of the foregoing first, second, third and fourth lipidnanoparticles, each KRAS activating oncogene mutation peptide comprises20, 21, 22, 23, 24, or 25 amino acids in length. In some aspects, eachKRAS activating oncogene mutation peptide comprises 25 amino acids inlength.

In some aspects of the foregoing first, second, third and fourth lipidnanoparticles, the KRAS activating oncogene mutation peptide comprisesthe amino acid sequences set forth in SEQ ID NO: 39. In some aspects,the mRNA encoding the KRAS activating oncogene mutation peptidecomprises the nucleotide sequence set forth in SEQ ID NOs: 126 or 190.

In some aspects of the foregoing first, second, third and fourth lipidnanoparticles, the KRAS activating oncogene mutation peptide comprisesthe amino acid sequences set forth in SEQ ID NO: 40. In some aspect, themRNA encoding the KRAS activating oncogene mutation peptide comprisesthe nucleotide sequence set forth in SEQ ID NOs: 127 or 191.

In some aspects of the foregoing first, second, third and fourth lipidnanoparticles, the KRAS activating oncogene mutation peptide comprisesthe amino acid sequences set forth in SEQ ID NO: 72. In some aspects,the mRNA encoding the KRAS activating oncogene mutation peptidecomprises the nucleotide sequence set forth in SEQ ID NOs: 132 or 196.

In some aspects of the foregoing first, second, third and fourth lipidnanoparticles, wherein the KRAS activating oncogene mutation peptidecomprises the amino acid sequences set forth in SEQ ID NO: 41. In someaspects, the mRNA encoding the KRAS activating oncogene mutation peptidecomprises the nucleotide sequence set forth in SEQ ID NOs: 128 or 192.

In some aspects of the foregoing first, second, third and fourth lipidnanoparticles, the constitutively active human STING polypeptidecomprises mutation V155M. In some aspects, the constitutively activehuman STING polypeptide comprises the amino acid sequence shown in SEQID NO: 1. In some aspects, the constitutively active human STINGpolypeptide comprises a 3′ UTR comprising at least one miR-122 microRNAbinding site. In some aspects, the mRNA encoding the constitutivelyactive human STING polypeptide comprises the nucleotide sequence shownin SEQ ID NO: 139, SEQ ID NO: 168, or SEQ ID NO: 170.

In some aspects, the disclosure provides a drug product comprising anyof the foregoing or related lipid nanoparticles for use in cancertherapy, optionally with instructions for use in cancer therapy. In someaspects, the disclosure provides a drug product comprising any of theforegoing first, second, third and fourth lipid nanoparticles, for usein cancer therapy, optionally with instructions for use in cancertherapy.

In some aspects, the disclosure provides a drug product comprising afirst, second, third and fourth lipid nanoparticles, for use in cancertherapy, optionally with instructions for use in cancer therapy,wherein:

(i) the first lipid nanoparticle comprises: an mRNA comprising an openreading frame encoding a KRAS activating oncogene mutation peptidecomprising G12D; and an mRNA comprising an open reading frame encoding aconstitutively active human STING polypeptide;

(ii) the second lipid nanoparticle comprises: an mRNA comprising an openreading frame encoding a KRAS activating oncogene mutation peptidecomprising G12V; and an mRNA comprising an open reading frame encoding aconstitutively active human STING polypeptide;

(iii) the third lipid nanoparticle comprises: an mRNA comprising an openreading frame encoding a KRAS activating oncogene mutation peptidecomprising G12C; and an mRNA comprising an open reading frame encoding aconstitutively active human STING polypeptide; and

(iv) the fourth lipid nanoparticle comprises: an mRNA comprising an openreading frame encoding a KRAS activating oncogene mutation peptidecomprising G13D; and an mRNA comprising an open reading frame encoding aconstitutively active human STING polypeptide.

In any of the foregoing or related aspects, the disclosure provides amethod for treating a subject, comprising: administering to a subjecthaving cancer any of the foregoing or related immunomodulatorytherapeutic compositions or any of the foregoing or related lipidnanoparticle. In some aspects, the immunomodulatory therapeuticcomposition or lipid nanoparticle is administered in combination with acancer therapeutic agent. In some aspects, the immunomodulatorytherapeutic composition or lipid nanoparticle is administered incombination with an inhibitory checkpoint polypeptide. In some aspects,the inhibitory checkpoint polypeptide is an antibody or fragment thereofthat specifically binds to a molecule selected from the group consistingof PD-1, PD-L1, TIM-3, VISTA, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIRand LAG3.

Methods provided herein may be used for treating a subject havingcancer. In some embodiments, the cancer is selected from cancer of thepancreas, peritoneum, large intestine, small intestine, biliary tract,lung, endometrium, ovary, genital tract, gastrointestinal tract, cervix,stomach, urinary tract, colon, rectum, and hematopoietic and lymphoidtissues. In some embodiments, the cancer is colorectal cancer. In someembodiments, the cancer is pancreatic cancer. In some embodiments, thecancer is lung cancer, such as non-small cell lung cancer (NSCLC). Insome embodiments, the cancer is selected from the group consisting ofcolorectal cancer, pancreatic cancer and lung cancer (e.g., NSCLC).

An mRNA (e.g., mmRNA) construct of the disclosure (e.g., an immunepotentiator mRNA, antigen-encoding mRNA, or combination thereof) cancomprise, for example, a 5′ UTR, a codon optimized open reading frameencoding the polypeptide, a 3′ UTR and a 3′ tailing region of linkednucleosides. In one embodiment, the mRNA further comprises one or moremicroRNA (miRNA) binding sites.

In one embodiment, a modified mRNA construct of the disclosure is fullymodified. For example, in one embodiment, the mmRNA comprisespseudouridine (ψ), pseudouridine (ψ) and 5-methyl-cytidine (m⁵C),1-methyl-pseudouridine (m¹ψ), 1-methyl-pseudouridine (m¹ψ) and5-methyl-cytidine (m⁵C), 2-thiouridine (s²U), 2-thiouridine and5-methyl-cytidine (m⁵C), 5-methoxy-uridine (mo⁵U), 5-methoxy-uridine(mo⁵U) and 5-methyl-cytidine (m⁵C), 2′-O-methyl uridine, 2′-O-methyluridine and 5-methyl-cytidine (m⁵C), N6-methyl-adenosine (m⁶A) orN6-methyl-adenosine (m⁶A) and 5-methyl-cytidine (m⁵C). In anotherembodiment, the mmRNA comprises pseudouridine (ψ),N1-methylpseudouridine (m¹ψ), 2-thiouridine, 4′-thiouridine,5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine,2-thio-dihydropseudouridine, 2-thio-dihydrouridine,2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine,4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine,4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine,5-methoxyuridine, or 2′-O-methyl uridine, or combinations thereof. Inyet another embodiment, the mmRNA comprises 1-methyl-pseudouridine(m¹ψ), 5-methoxy-uridine (mo⁵U), 5-methyl-cytidine (m⁵C), pseudouridine(ψ), α-thio-guanosine, or α-thio-adenosine, or combinations thereof. Insome aspects, the mmRNA comprises pseudouridine or a pseudouridineanalog. In some aspects, the mmRNA comprises N1-methylpseudouridine. Insome aspects, each mmRNA comprises fully modifiedN1-methylpseudouridine.

In some embodiments the dosage of the RNA polynucleotide in theimmunomodulatory therapeutic composition is 1-5 μg, 5-10 μg, 10-15 μg,15-20 μg, 10-25 μg, 20-25 μg, 20-50 μg, 30-50 μg, 40-50 μg, 40-60 μg,60-80 μg, 60-100 μg, 50-100 μg, 80-120 μg, 40-120 μg, 40-150 μg, 50-150μg, 50-200 μg, 80-200 μg, 100-200 μg, 100-300 μg, 120-250 μg, 150-250μg, 180-280 μg, 200-300 μg, 30-300 μg, 50-300 μg, 80-300 μg, 100-300 μg,40-300 μg, 50-350 μg, 100-350 μg, 200-350 μg, 300-350 μg, 320-400 μg,40-380 g, 40-100 μg, 100-400 μg, 200-400 μg, or 300-400 μg per dose. Insome embodiments, the immunomodulatory therapeutic composition isadministered to the subject by intradermal or intramuscular injection.In some embodiments, the immunomodulatory therapeutic composition isadministered to the subject on day zero. In some embodiments, a seconddose of the immunomodulatory therapeutic composition is administered tothe subject on day twenty one.

In some embodiments, a dosage of 25 micrograms of the RNA polynucleotideis included in the immunomodulatory therapeutic composition administeredto the subject. In some embodiments, a dosage of 10 micrograms of theRNA polynucleotide is included in the immunomodulatory therapeuticcomposition administered to the subject. In some embodiments, a dosageof 30 micrograms of the RNA polynucleotide is included in theimmunomodulatory therapeutic composition administered to the subject. Insome embodiments, a dosage of 100 micrograms of the RNA polynucleotideis included in the immunomodulatory therapeutic composition administeredto the subject. In some embodiments, a dosage of 50 micrograms of theRNA polynucleotide is included in the immunomodulatory therapeuticcomposition administered to the subject. In some embodiments, a dosageof 75 micrograms of the RNA polynucleotide is included in theimmunomodulatory therapeutic composition administered to the subject. Insome embodiments, a dosage of 150 micrograms of the RNA polynucleotideis included in the immunomodulatory therapeutic composition administeredto the subject. In some embodiments, a dosage of 400 micrograms of theRNA polynucleotide is included in the immunomodulatory therapeuticcomposition administered to the subject. In some embodiments, a dosageof 300 micrograms of the RNA polynucleotide is included in theimmunomodulatory therapeutic composition administered to the subject. Insome embodiments, a dosage of 200 micrograms of the RNA polynucleotideis included in the immunomodulatory therapeutic composition administeredto the subject. In some embodiments, the RNA polynucleotide accumulatesat a 100 fold higher level in the local lymph node in comparison withthe distal lymph node. In other embodiments the immunomodulatorytherapeutic composition is chemically modified and in other embodimentsthe immunomodulatory therapeutic composition is not chemically modified.

In some embodiments, the effective amount is a total dose of 1-100 μg.In some embodiments, the effective amount is a total dose of 100 μg. Insome embodiments, the effective amount is a dose of 25 μg administeredto the subject a total of one or two times. In some embodiments, theeffective amount is a dose of 100 μg administered to the subject a totalof two times. In some embodiments, the effective amount is a dose of 1μg-10 μg, 1 μg-20 μg, 1 μg-30 μg, 5 μg-10 μg, 5 μg-20 μg, 5 μg-30 μg, 5μg-40 μg, 5 μg-50 μg, 10 μg-15 μg, 10 μg-20 μg, 10 μg-25 μg, 10 μg-30μg, 10 μg-40 μg, 10 μg-50 μg, 10 μg-60 μg, 15 μg-20 μg, 15 μg-25 μg, 15μg-30 μg, 15 μg-40 μg, 15 μg-50 μg, 20 μg-25 μg, 20 μg-30 μg, 20 μg-40μg 20 μg-50 μg, 20 μg-60 μg, 20 μg-70 μg, 20 μg-75 μg, 30 μg-35 μg, 30μg-40 μg, 30 μg-45 μg 30 μg-50 μg, 30 μg-60 μg, 30 μg-70 μg, 30 μg-75 μgwhich may be administered to the subject a total of one or two times ormore.

In some aspects, the disclosure provides a composition (e.g., a vaccine)comprising an mRNA encoding a KRAS activating oncogene mutation peptideand an mRNA encoding a constitutively active human STING polypeptidewherein the mRNA encoding the KRAS activating oncogene mutation peptideand the mRNA encoding the constitutively active human STING polypeptideare present at a KRAS:STING mass ratio of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1,7:1, 8:1, 9:1, 10:1 or 20:1, or alternatively at a STING:KRAS mass ratioof 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10 or 1:20. In someaspects, the mRNAs are present at a mass ratio of 5:1 of mRNA encodingthe KRAS activating oncogene mutation peptide to the mRNA encoding theconstitutively active human STING polypeptide (KRAS:STING mass ratio of5:1 or alternatively a STING:KRAS mass ratio of 1:5). In some aspects,the mRNAs are present at a mass ratio of 10:1 of mRNA encoding the KRASactivating oncogene mutation peptide to the mRNA encoding theconstitutively active human STING polypeptide (KRAS:STING mass ratio of10:1 or alternatively a STING:KRAS ratio of 1:10).

Other aspects of the disclosure relate to a lipid nanoparticlecomprising:

an mRNA comprising an open reading frame encoding a concatemer of 4 KRASactivating oncogene mutation peptides, wherein the 4 KRAS activatingoncogene mutation peptides comprise G12D, G12V, G12C, and G13D;

an mRNA comprising an open reading frame encoding a constitutivelyactive human STING polypeptide;

wherein the mRNAs are present at a KRAS:STING mass ratio selected fromthe group consisting of of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1or 10:1.

In some aspects, the disclosure relates to a lipid nanoparticlecomprising:

a first mRNAs comprising an open reading frame encoding a KRASactivating oncogene mutation peptide comprising G12D;

a second mRNA comprising an open reading frame encoding a KRASactivating oncogene mutation peptide comprising G12V;

a third mRNA comprising an open reading frame encoding a KRAS activatingoncogene mutation peptide comprising G12C;

a fourth mRNA comprising an open reading frame encoding a KRASactivating oncogene mutation peptide comprising G13D;

a fifth mRNA comprising an open reading frame encoding a constitutivelyactive human STING polypeptide;

wherein the first, second, third, fourth and fifth mRNAs are present atan KRAS:STING mass ratio selected from the group consisting of of 1:1,2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1.

In some of the foregoing and related aspects, the concatemer comprisesfrom N- to C-terminus G12D, G12V, G13D, and G12C. In some aspects, theconcatemer comprises from N- to C-terminus G12C, G13D, G12V, and G12D.In some aspects, each KRAS activating oncogene mutation peptidecomprises 20, 21, 22, 23, 24, or 25 amino acids in length. In someaspects, each KRAS activating oncogene mutation peptide comprises 25amino acids in length. In some aspects, the concatemer comprises anamino acid sequence set forth in SEQ ID NO: 137. In some aspects, themRNA encoding the concatemer of 4 KRAS activating oncogene mutationpeptides comprises the nucleotide sequence set forth in SEQ ID NO: 138,SEQ ID NO: 167 or SEQ ID NO: 169. In some aspects, the constitutivelyactive human STING polypeptide comprises mutation V155M. In someaspects, the constitutively active human STING polypeptide comprises theamino acid sequence shown in SEQ ID NO: 1. In some aspects, the mRNAencoding the constitutively active human STING polypeptide comprises a3′ UTR comprising at least one miR-122 microRNA binding site. In someaspects, the mRNA encoding the constitutively active human STINGpolypeptide comprises the nucleotide sequence shown in SEQ ID NO: 139,SEQ ID NO: 168, or SEQ ID NO: 170.

In some of the foregoing and related aspects, the lipid nanoparticlecomprises mRNAs present at an KRAS:STING mass ratio of 1:1. In someaspects, the mRNAs are present at a KRAS:STING mass ratio of 2:1. Insome aspects, the mRNAs are present at a KRAS:STING mass ratio of 3:1.In some aspects, the the mRNAs are present at a KRAS:STING mass ratio of4:1. In some aspects, the mRNAs are present at a KRAS:STING mass ratioof 5:1. In some aspects, the mRNAs are present at a KRAS:STING massratio of 6:1. In some aspects, the mRNAs are present at a KRAS:STINGmass ratio of 7:1. In some aspects, the mRNAs are present at aKRAS:STING mass ratio of 8:1. In some aspects, the mRNAs are present ata KRAS:STING mass ratio of 9:1. In some aspects, the mRNAS are presentat a KRAS:STING mass ratio of 10:1.

In another aspect, the disclosure pertains to a lipid nanoparticlecomprising a modified mRNA of the disclosure. In one embodiment, thelipid nanoparticle is a liposome. In another embodiment, the lipidnanoparticle comprises a cationic and/or ionizable amino lipid. In oneembodiment, the cationic and/or ionizable amino lipid is2,2-dilinoleyl-4-methylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA) ordilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA). In someaspects, the ionizable amino lipid comprises a compound of any ofFormulae (I), (IA), (II), (IIa), (IIb), (IIc), (IId), and (IIe). In someaspects, the ionizable amino lipid comprises a compound of Formula (I).In one embodiment, the ionizable amino lipid is Compound 25. In oneembodiment, the lipid nanoparticle further comprises a targeting moietyconjugated to the outer surface of the lipid nanoparticle.

In another aspect, the disclosure pertains to a pharmaceuticalcomposition comprising a modified mRNA of the disclosure or a lipidnanoparticle of the disclosure, and a pharmaceutically acceptablecarrier, diluent or excipient.

In another aspect, the disclosure pertains to a method for enhancing animmune response to an antigen(s) of interest, the method comprisingadministering to a subject in need thereof a mRNA composition ofdisclosure encoding an antigen(s) of interest and a polypeptide thatenhances an immune response to the antigen(s) of interest, or lipidnanoparticle thereof, or pharmaceutical composition thereof, such thatan immune response to the antigen of interest is enhanced in thesubject. In one aspect, enhancing an immune response in a subjectcomprises stimulating cytokine production (e.g., IFN-γ or TNF-α). Inanother aspect, enhancing an immune response in a subject comprisesstimulating antigen-specific CD8⁺ T cell activity, e.g., priming,proliferation and/or survival (e.g., increasing the effector/memory Tcell population). In one aspect, enhancing an immune response in asubject comprises stimulating antigen-specific CD4⁺ T cell activity(e.g., increasing helper T cell activity). In other aspects, enhancingan immune response in a subject comprises stimulating B cell responses(e.g., increasing antibody production).

In one aspect, the disclosure provides methods for enhancing an immuneresponse to an activating oncogene mutation peptide, wherein the subjectis administered two different immune potentiator mRNA (e.g., mmRNA)constructs (wherein one or both constructs also encode, or areadministered with an mRNA (e.g., mmRNA) construct that encodes, theactivating oncogene mutation peptide), either at the same time orsequentially. In one aspect, the subject is administered an immunepotentiator mmRNA composition that stimulates dendritic cell developmentor activity prior to administering to the subject an immune potentiatormRNA composition that stimulates Type I interferon pathway signaling.

In other aspects, the disclosure provides methods of stimulating animmune response to a tumor in a subject in need thereof, wherein themethod comprises administering to the subject an effective amount of acomposition comprising at least one mRNA construct encoding a tumorantigen(s) and an mRNA construct encoding a polypeptide that enhances animmune response to the tumor antigen(s), or a lipid nanoparticlethereof, or a pharmaceutical composition thereof, such that an immuneresponse to the tumor is stimulated in the subject. In one aspect, thetumor is a liver cancer, a colorectal cancer, a pancreatic cancer, anon-small cell lung cancer (NSCLC), a melanoma cancer, a cervical canceror a head or neck cancer.

In another aspect, the disclosure provides a composition comprising:

(i) a first mRNA comprising an open reading frame encoding a concatemerof 4 KRAS activating oncogene mutation peptides, wherein the concatemercomprises from N- to C-terminus G12D, G12V, G13D, and G12C, and

(ii) a second mRNA comprising an open reading frame encoding aconstitutively active human STING polypeptide, wherein theconstitutively active human STING polypeptide comprises mutation V155M,

wherein the first mRNA and second mRNA are present at a KRAS:STING massratio selected from the group consisting of 1:1, 2:1, 3:1, 4:1, 5:1,6:1, 7:1, 8:1, 9:1 or 10:1;

and a pharmaceutically acceptable carrier.

In some aspects of the foregoing composition, the concatemer of 4 KRASactivating oncogene mutation peptides comprises the amino acid sequenceset forth in SEQ ID NO: 137. In some aspects, the first mRNA encodingthe concatemer of 4 KRAS activating oncogene mutation peptides comprisesthe nucleotide sequence set forth in SEQ ID NO: 169. In some aspects,the constitutively active human STING polypeptide comprises the aminoacid sequence shown in SEQ ID NO: 1. In some aspects, the mRNA encodingthe constitutively active human STING polypeptide comprises thenucleotide sequence shown in SEQ ID NO: 170. In some aspects, the firstmRNA comprises a 5′ UTR comprising the nucleotide sequence set forth inSEQ ID NO: 176. In some aspects, the second mRNA comprises a 5′ UTRcomprising the nucleotide sequence set forth in SEQ ID NO: 176. In someaspects, the second mRNA encoding the constitutively active human STINGpolypeptide comprises a 3′ UTR having a miR-122 microRNA binding site.In some aspects, the miR-122 microRNA binding site comprises thenucleotide sequence shown in SEQ ID NO: 175. In some aspects, the firstmRNA and second mRNA each comprise a poly A tail. In some aspects, thepoly A tail comprises about 100 nucleotides. In some aspects, the firstand second mRNAs each comprise a 5′ Cap 1 structure. In some aspects,the first and second mRNAs each comprise at least one chemicalmodification. In some aspects, the chemical modification isN1-methylpseudouridine. In some aspects, the first mRNA is fullymodified with N1-methylpseudouridine. In some aspects, the second mRNAis fully modified with N1-methylpseudouridine. In some aspects, thepharmaceutically acceptable carrier comprises a buffer solution.

In another aspect, the disclosure provides a composition comprising:

(i) a first mRNA comprising the nucleotide sequence set forth in SEQ IDNO: 167, and

(ii) a second mRNA comprising the nucleotide sequence set forth in SEQID NO: 168,

wherein the first and second mRNA are each fully modified withN1-methylpseudouridine, and

wherein the first mRNA and second mRNA are present at a KRAS:STING massratio selected from the group consisting of 1:1, 2:1, 3:1, 4:1, 5:1,6:1, 7:1, 8:1, 9:1 or 10:1; and a pharmaceutically acceptable carrier.

In one aspect of the foregoing composition, the pharmaceuticallyacceptable carrier comprises a buffer solution.

In any of the foregoing or related aspects, the disclosure provides acomposition wherein the first and second mRNAs are present at aKRAS:STING mass ratio of 1:1.

In any of the foregoing or related aspects, the disclosure provides acomposition wherein the first and second mRNAs are present at aKRAS:STING mass ratio of 2:1.

In any of the foregoing or related aspects, the disclosure provides acomposition wherein the first and second mRNAs are present at aKRAS:STING mass ratio of 3:1.

In any of the foregoing or related aspects, the disclosure provides acomposition wherein the first and second mRNAs are present at aKRAS:STING mass ratio of 4:1.

In any of the foregoing or related aspects, the disclosure provides acomposition wherein the first and second mRNAs are present at aKRAS:STING mass ratio of 5:1.

In any of the foregoing or related aspects, the disclosure provides acomposition wherein the first and second mRNAs are present KRAS:STINGmass ratio of 6:1.

In any of the foregoing or related aspects, the disclosure provides acomposition wherein the first and second mRNAs are present at aKRAS:STING mass ratio of 7:1.

In any of the foregoing or related aspects, the disclosure provides acomposition wherein the first and second mRNAs are present at aKRAS:STING mass ratio of 8:1.

In any of the foregoing or related aspects, the disclosure provides acomposition wherein the first and second mRNAs are present at aKRAS:STING mass ratio of 9:1.

In any of the foregoing or related aspects, the disclosure provides acomposition wherein the first and second mRNAs are present at aKRAS:STING mass ratio of 10:1.

In any of the foregoing or related aspects, the disclosure provides acomposition which is formulated in a lipid nanoparticle. In someaspects, the lipid nanoparticle comprises a molar ratio of about 20-60%ionizable amino lipid:5-25% phospholipid:25-55% sterol; and 0.5-15%PEG-modified lipid. In some aspects, the lipid nanoparticle comprises amolar ratio of about 50% Compound 25:about 10% DSPC:about 38.5%cholesterol; and about 1.5% PEG-DMG.

In any of the foregoing or related aspects, the disclosure provides acomposition which is formulated for intramuscular delivery.

In some aspects, the disclosure provides a lipid nanoparticlecomprising:

(i) a first mRNA comprising an open reading frame encoding a concatemerof 4 KRAS activating oncogene mutation peptides, wherein the concatemercomprises from N- to C-terminus G12D, G12V, G13D, and G12C; and

(ii) a second mRNA comprising an open reading frame encoding aconstitutively active human STING polypeptide, wherein theconstitutively active human STING polypeptide comprises mutation V155M,

wherein the first mRNA and second mRNA are present at a KRAS:STING massratio of 5:1.

In some aspects of the foregoing lipid nanoparticle, the concatemer of 4KRAS activating oncogene mutation peptides comprises the amino acidsequence set forth in SEQ ID NO: 137. In some aspects, the first mRNAencoding the concatemer of 4 KRAS activating oncogene mutation peptidescomprises the nucleotide sequence set forth in SEQ ID NO: 169. In someaspects, the constitutively active human STING polypeptide comprises theamino acid sequence shown in SEQ ID NO: 1. In some aspects, the mRNAencoding the constitutively active human STING polypeptide comprises thenucleotide sequence shown in SEQ ID NO: 170. In some aspects, the firstmRNA comprises a 5′ UTR comprising the nucleotide sequence shown in SEQID NO: 176. In some aspects, the second mRNA comprises a 5′ UTRcomprising the nucleotide sequence shown in SEQ ID NO: 176. In someaspects, the second mRNA encoding the constitutively active human STINGpolypeptide comprises a 3′ UTR having a miR-122 microRNA binding site.In some aspects, the miR-122 microRNA binding site comprises thenucleotide sequence shown in SEQ ID NO: 175. In some aspects, the firstand second mRNAs each comprise a poly A tail. In some aspects, the polyA tail comprises about 100 nucleotides. In some aspects, the first andsecond mRNAs each comprise a 5′ Cap 1 structure. In some aspects, thefirst and second mRNAs each comprise at least one chemical modification.In some aspects, the chemical modification is N1-methylpseudouridine. Insome aspects, the first mRNA is fully modified withN1-methylpseudouridine. In some aspects, the second mRNA is fullymodified with N1-methylpseudouridine.

In some aspects, the disclosure provides a lipid nanoparticlecomprising:

(i) a first mRNA comprising the nucleotide sequence set forth in SEQ IDNO: 167; and

(ii) a second mRNA comprising the nucleotide sequence set forth in SEQID NO: 168,

wherein the first and second mRNA are each fully modified withN1-methylpseudouridine, and

wherein the first mRNA and second mRNA are present at a KRAS:STING massratio of 5:1.

In some aspects of the foregoing lipid nanoparticle, the lipidnanoparticle comprises a molar ratio of about 20-60% ionizable aminolipid:5-25% phospholipid:25-55% sterol; and 0.5-15% PEG-modified lipid.In some aspects, the ionizable amino lipid comprises a compound of anyof Formulae (I), (IA), (II), (IIa), (IIb), (IIc), (IId), and (IIe). Insome aspects, the ionizable amino lipid comprises a compound of Formula(I). In some aspects, the compound of Formula (I) is Compound 25. Insome aspects, the lipid nanoparticle comprises a molar ratio of about50% Compound 25:about 10% DSPC:about 38.5% cholesterol; and about 1.5%PEG-DMG.

In any of the foregoing or related aspects, the disclosure providespharmaceutical composition comprising the lipid nanoparticle, and apharmaceutically acceptable carrier. In some aspects, the pharmaceuticalcomposition is formulated for intramuscular delivery.

In any of the foregoing or related aspects, the disclosure provides alipid nanoparticle, and an optional pharmaceutically acceptable carrier,or a pharmaceutical composition for use in treating or delayingprogression of cancer in an individual, wherein the treatment comprisesadministration of the composition in combination with a secondcomposition, wherein the second composition comprises a checkpointinhibitor polypeptide and an optional pharmaceutically acceptablecarrier.

In any of the foregoing or related aspects, the disclosure provides useof a lipid nanoparticle, and an optional pharmaceutically acceptablecarrier, in the manufacture of a medicament for treating or delayingprogression of cancer in an individual, wherein the medicament comprisesthe lipid nanoparticle and an optional pharmaceutically acceptablecarrier and wherein the treatment comprises administration of themedicament in combination with a composition comprising a checkpointinhibitor polypeptide and an optional pharmaceutically acceptablecarrier.

In any of the foregoing or related aspects, the disclosure provides akit comprising a container comprising a lipid nanoparticle, and anoptional pharmaceutically acceptable carrier, or a pharmaceuticalcomposition, and a package insert comprising instructions foradministration of the lipid nanoparticle or pharmaceutical compositionfor treating or delaying progression of cancer in an individual. In someaspects, the package insert further comprises instructions foradministration of the lipid nanoparticle or pharmaceutical compositionin combination with a composition comprising a checkpoint inhibitorpolypeptide and an optional pharmaceutically acceptable carrier fortreating or delaying progression of cancer in an individual.

In any of the foregoing or related aspects, the disclosure provides akit comprising a medicament comprising a lipid nanoparticle, and anoptional pharmaceutically acceptable carrier, or a pharmaceuticalcomposition, and a package insert comprising instructions foradministration of the medicament alone or in combination with acomposition comprising a checkpoint inhibitor polypeptide and anoptional pharmaceutically acceptable carrier for treating or delayingprogression of cancer in an individual. In some aspects, the kit furthercomprises a package insert comprising instructions for administration ofthe first medicament prior to, current with, or subsequent toadministration of the second medicament for treating or delayingprogression of cancer in an individual.

In any of the foregoing or related aspects, the disclosure provides alipid nanoparticle, a composition, or the use thereof, or a kitcomprising a lipid nanoparticle or a composition as described herein,wherein the checkpoint inhibitor polypeptide inhibits PD1, PD-L1, CTLA4,or a combination thereof. In some aspects, the checkpoint inhibitorpolypeptide is an antibody. In some aspects, the checkpoint inhibitorpolypeptide is an antibody selected from an anti-CTLA4 antibody orantigen-binding fragment thereof that specifically binds CTLA4, ananti-PD1 antibody or antigen-binding fragment thereof that specificallybinds PD1, an anti-PD-L1 antibody or antigen-binding fragment thereofthat specifically binds PD-L1, and a combination thereof. In someaspects, the checkpoint inhibitor polypeptide is an anti-PD-L1 antibodyselected from atezolizumab, avelumab, or durvalumab. In some aspects,the checkpoint inhibitor polypeptide is an anti-CTLA-4 antibody selectedfrom tremelimumab or ipilimumab. In some aspects, the checkpointinhibitor polypeptide is an anti-PD1 antibody selected from nivolumab orpembrolizumab. In some asepcts, the checkpoint inhibitor polypeptide isan anti-PD1 antibody, wherein the anti-PD1 antibody is pembrolizumab.

In related aspects, the disclosure provides a method of reducing ordecreasing a size of a tumor or inhibiting a tumor growth in a subjectin need thereof comprising administering to the subject any of theforegoing or related lipid nanoparticles of the disclosure, or any ofthe foregoing or related compositions of the disclosure.

In related aspects, the disclosure provides a method inducing ananti-tumor response in a subject with cancer comprising administering tothe subject any of the foregoing or related lipid nanoparticles of thedisclosure, or any of the foregoing or related compositions of thedisclosure. In some aspects, the anti-tumor response comprises a T-cellresponse. In some aspects, the T-cell response comprises CD8+ T cells.

In some aspects of the foregoing methods, the composition isadministered by intramuscular injection.

In some aspects of the foregoing methods, the method further comprisesadministering a second composition comprising a checkpoint inhibitorpolypeptide, and an optional pharmaceutically acceptable carrier. Insome aspects, the checkpoint inhibitor polypeptide inhibits PD1, PD-L,CTLA4, or a combination thereof. In some aspects, the checkpointinhibitor polypeptide is an antibody. In some aspects, the checkpointinhibitor polypeptide is an antibody selected from an anti-CTLA4antibody or antigen-binding fragment thereof that specifically bindsCTLA4, an anti-PD1 antibody or antigen-binding fragment thereof thatspecifically binds PD1, an anti-PD-L1 antibody or antigen-bindingfragment thereof that specifically binds PD-L1, and a combinationthereof. In some aspects, the checkpoint inhibitor polypeptide is ananti-PD-L1 antibody selected from atezolizumab, avelumab, or durvalumab.In some aspects, the checkpoint inhibitor polypeptide is an anti-CTLA-4antibody selected from tremelimumab or ipilimumab. In some aspects, thecheckpoint inhibitor polypeptide is an anti-PD1 antibody selected fromnivolumab or pembrolizumab. In some asepcts, the checkpoint inhibitorpolypeptide is an anti-PD1 antibody, wherein the anti-PD1 antibody ispembrolizumab.

In some aspects of any of the foregoing or related methods, thecomposition comprising the checkpoint inhibitor polypeptide isadministered by intravenous injection. In some aspects, the compositioncomprising the checkpoint inhibitor polypeptide is administered onceevery 2 to 3 weeks. In some aspects, the composition comprising thecheckpoint inhibitor polypeptide is administered once every 2 weeks oronce every 3 weeks. In some aspects, the composition comprising thecheckpoint inhibitor polypeptide is administered prior to, concurrentwith, or subsequent to administration of the lipid nanoparticle orpharmaceutical composition thereof.

In some aspects of any of the foregoing or related methods, the subjecthas a histologically confirmed KRAS mutation selected from G12D, G12V,G13D or G12C.

In some aspects of any of the foregoing or related methods, the subjecthas metastatic colorectal cancer.

In some aspects of any of the foregoing or related methods, the subjecthas non-small cell lung cancer (NSCLC).

In some aspects of any of the foregoing or related methods, the subjecthas pancreatic cancer

In other aspects, the disclosure provides a method of reducing ordecreasing a size of a tumor, inhibiting a tumor growth or inducing ananti-tumor response in a subject in need thereof, comprisingadministering to the subject an immunomodulatory therapeutic compositioncomprising: one or more first mRNA each comprising an open reading frameencoding a KRAS activating oncogene mutation peptide, and optionally oneor more second mRNA each comprising an open reading frame encoding aconstitutively active human STING polypeptide, and optionally whereinthe first mRNA and second mRNA are at a mass ratio selected from thegroup consisting of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1;and a pharmaceutically acceptable carrier, thereby reducing ordecreasing a size of a tumor, inhibiting a tumor growth or inducing ananti-tumor response in the subject. In some aspects, the compositioncomprises 1, 2, 3, or 4 mRNAs encoding 1, 2, 3, or 4 KRAS activatingoncogene mutation peptides. In some aspects, the composition comprises 4mRNAs encoding 4 KRAS activating oncogene mutation peptides. In someaspects, the 4 KRAS activating oncogene mutation peptides comprise G12D,G12V, G12C, and G13D.

In other aspects, the method comprises administering an immunomodulatorytherapeutic composition comprising a first, second, third, fourth, andfifth mRNA, wherein

the first mRNA comprises an open reading frame encoding a KRASactivating oncogene mutation peptide comprising G12D;

the second mRNA comprises an open reading frame encoding a KRASactivating oncogene mutation peptide comprises G12V;

the third mRNA comprises an open reading frame encoding a KRASactivating oncogene mutation peptide comprising G12C;

the fourth mRNA comprises an open reading frame encoding a KRASactivating oncogene mutation peptide comprising G13D; and

the fifth mRNA comprises an open reading frame encoding a constitutivelyactive human STING polypeptide,

wherein the first, second, third, fourth and fifth mRNAs are present ata KRAS:STING mass ratio selected from the group consisting of 1:1, 2:1,3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1.

In some aspects, KRAS activating oncogene mutation peptides comprise theamino acid sequences set forth in SEQ ID NOs: 39-41 and 72. In someaspects, the mRNA encoding the KRAS activating oncogene mutation peptidecomprises the nucleotide sequences set forth in SEQ ID NOs: 126-128 and132.

In other aspects, the method comprises administering an immunomodulatorytherapeutic composition comprising an mRNA comprising an open readingframe encoding a concatemer of two or more KRAS activating oncogenemutation peptides. In some aspects, the concatemer comprises G12D, G12V,G12C, and G13D. In some aspects, the concatemer comprises from N- toC-terminus G12D, G12V, G13D, and G12C. In some aspects, the concatemercomprises from N- to C-terminus G12C, G13D, G12V, and G12D. In someaspects, the concatemer comprises an amino acid sequence selected fromthe group set forth in SEQ ID NOs: 42-47, 73 and 137. In some aspects,the mRNA encoding the concatemer comprises the nucleotide sequenceselected from the group set forth in SEQ ID NOs: 129-131, 133 and 138.

In some aspects, the disclosure provides a method of reducing ordecreasing a size of a tumor, inhibiting a tumor growth or inducing ananti-tumor response in a subject in need thereof, comprisingadministering to the subject a lipid nanoparticle comprising:

-   -   (i) one or more first mRNAs selected from the group consisting        of:        -   (a) an mRNA comprising an open reading frame encoding a KRAS            activating oncogene mutation peptide comprising G12D;        -   (b) an mRNA comprising an open reading frame encoding a KRAS            activating oncogene mutation peptide comprising G12V;        -   (c) an mRNA comprising an open reading frame encoding a KRAS            activating oncogene mutation peptide comprising G12C;        -   (d) an mRNA comprising an open reading frame encoding a KRAS            activating oncogene mutation peptide comprising G13D;        -   (e) an mRNA comprising an open reading frame encoding a            concatemer of 2, 3, or 4 KRAS activating oncogene mutation            peptides, wherein the KRAS activating oncogene mutation            peptides comprise G12D, G12V, G12C, and G13D; and        -   (f) any combination of mRNAs set forth in (a)-(d); and    -   (ii) one or more second mRNAs each comprising an open reading        frame encoding a constitutively active human STING polypeptide,        optionally

wherein the first mRNA and second mRNA are present at a KRAS:STING massratio selected from the group consisting of 1:1, 2:1, 3:1, 4:1, 5:1,6:1, 7:1, 8:1, 9:1 or 10:1,

thereby reducing or decreasing a size of a tumor, inhibiting a tumorgrowth or inducing an anti-tumor response in the subject.

In some aspects, the lipid nanoparticle comprises

(i) a combination of mRNAs set forth in (a)-(d); and

(ii) a second mRNA comprising an open reading frame encoding aconstitutively active human STING polypeptide, wherein theconstitutively active human STING polypeptide comprises mutation V155M,

wherein the first mRNA and second mRNA are present at a KRAS:STING massratio selected from the group consisting of 1:1, 2:1, 3:1, 4:1, 5:1,6:1, 7:1, 8:1, 9:1 or 10:1.

In some aspects, the lipid nanoparticle comprises

(i) a first mRNA comprises an open reading frame encoding a concatemerof 4 KRAS activating oncogene mutation peptides, wherein the concatemercomprises from N- to C-terminus G12D, G12V, G13D, and G12C; and

(ii) a second mRNA comprising an open reading frame encoding aconstitutively active human STING polypeptide, wherein theconstitutively active human STING polypeptide comprises mutation V155M,

wherein the first mRNA and second mRNA are present at a KRAS:STING massratio selected from the group consisting of 1:1, 2:1, 3:1, 4:1, 5:1,6:1, 7:1, 8:1, 9:1 or 10:1.

In some aspects, the disclosure provides a method of reducing ordecreasing a size of a tumor, inhibiting a tumor growth or inducing ananti-tumor response in a subject in need thereof, comprisingadministering to the subject a lipid nanoparticle comprising:

(i) a first mRNA comprising the nucleotide sequence set forth in SEQ IDNO: 167; and

(ii) a second mRNA comprising the nucleotide sequence set forth in SEQID NO: 168,

wherein the first and second mRNA are each fully modified withN1-methylpseudouridine, and wherein the first mRNA and second mRNA arepresent at a mass ratio of 5:1. In some aspects, the lipid nanoparticlecomprises a molar ratio of about 50% Compound 25:about 10% DSPC:about38.5% cholesterol; and about 1.5% PEG-DMG.

In some aspects, the lipid nanoparticle or composition is administeredby intramuscular injection.

In some aspects, the anti-tumor response comprises a T-cell response,such as a CD8+ T cell response.

In some aspects, the disclosure provides a method of reducing ordecreasing a size of a tumor, inhibiting a tumor growth or inducing ananti-tumor response in a subject in need thereof, comprisingadministering to the subject an immunomodulatory therapeutic compositionor lipid nanoparticle of the disclosure in combination with (prior to,concurrent with or consecutively) a second composition comprising acheckpoint inhibitor polypeptide or polynucleotide encoding the same,and an optional pharmaceutically acceptable carrier. In some aspects,the checkpoint inhibitor polypeptide inhibits PD1, PD-L1, CTLA4, or acombination thereof. In some aspects,

the checkpoint inhibitor polypeptide is an antibody. In some aspects,the checkpoint inhibitor polypeptide is an antibody selected from ananti-CTLA4 antibody or antigen-binding fragment thereof thatspecifically binds CTLA4, an anti-PD1 antibody or antigen-bindingfragment thereof that specifically binds PD1, an anti-PD-L1 antibody orantigen-binding fragment thereof that specifically binds PD-L1, and acombination thereof. In some aspects, the checkpoint inhibitorpolypeptide is an anti-PD-L1 antibody selected from atezolizumab,avelumab, or durvalumab. In some aspects, the checkpoint inhibitorpolypeptide is an anti-CTLA-4 antibody selected from tremelimumab oripilimumab. In some aspects, the checkpoint inhibitor polypeptide is ananti-PD1 antibody selected from nivolumab or pembrolizumab.

In some aspects, the composition comprising the checkpoint inhibitorpolypeptide is administered by intravenous injection. In some aspects,the composition comprising the checkpoint inhibitor polypeptide isadministered once every 2 to 3 weeks. In some aspects, the compositioncomprising the checkpoint inhibitor polypeptide is administered onceevery 2 weeks or once every 3 weeks. In some aspects, the compositioncomprising the checkpoint inhibitor polypeptide is administered priorto, concurrent with, or subsequent to administration of the lipidnanoparticle or composition.

In some aspects, the disclosure provides methods for treating subjectshaving a histologically confirmed KRAS mutation selected from G12D,G12V, G13D or G12C. In some aspects, the subject has a histologicallyconfirmed HLA subtype selected from HLA-A11 and/or HLA-C*08.

In some aspects, wherein the tumor is metastatic colorectal cancer. Insome aspects, the tumor is non-small cell lung cancer (NSCLC). In someaspects, the tumor is pancreatic cancer.

In some aspects, the subject is administered a chemotherapeutic agentprior to, concurrent with, or subsequent to administration of the lipidnanoparticle or composition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph showing stimulation of IFN-β production in TF1acells transfected with constitutively active STING mmRNA constructs.

FIG. 2 is a bar graph showing activation of an interferon-sensitiveresponse element (ISRE) by constitutively active STING constructs. STINGvariants 23a and 23b correspond to SEQ ID NO: 1, STING variant 42corresponds to SEQ ID NO: 2, STING variants 19, 21a and 21b correspondto SEQ ID NO: 3, STING variant 41 corresponds to SEQ ID NO: 4, STINGvariant 43 corresponds to SEQ ID NO: 5, STING variant 45 corresponds toSEQ ID NO: 6, STING variant 46 corresponds to SEQ ID NO: 7, STINGvariant 47 corresponds to SEQ ID NO: 8, STING variant 56 corresponds toSEQ ID NO: 9 and STING variant 57 corresponds to SEQ ID NO: 10.

FIGS. 3A-3B are bar graphs showing activation of an interferon-sensitiveresponse element (ISRE) by constitutively active IRF3 constructs (FIG.3A) or constitutively active IRF7 constructs (FIG. 3B). IRF3 variants 1,3 and 4 correspond to SEQ ID NO: 12 and IRF3 variants 2 and 5 correspondto SEQ ID NO: 11 (variants have different tags). IRF7 variant 36corresponds to SEQ ID NO: 18 and variant 31 is the murine version of SEQID NO: 18. IRF7 variant 32 corresponds to SEQ ID NO: 17 and IRF7 variant33 corresponds to SEQ ID NO: 14.

FIG. 4 is a bar graph showing activation of an NFκB-luciferase reportergene by constitutively active cFLIP and IKKβ mRNA constructs.

FIG. 5 is a graph showing activation of an NFκB-luciferase reporter geneby constitutively active RIPK1 mRNA constructs.

FIG. 6 is a bar graph showing TNF-α induction in SKOV3 cells transfectedwith DIABLO mmRNA constructs.

FIG. 7 is a bar graph showing interleukin 6 (IL-6) induction in SKOV3cells transfected with DIABLO mmRNA constructs.

FIGS. 8A-8B are graphs showing MC38 antigen-specific responses by IFN-γintracellular staining (ICS) of day 21 (FIG. 8A) or day 35 (FIG. 8B)CD8⁺ spenocytes from mice immunized with MC38 neo-antigen vaccineconstruct (ADRvax) coformulated with either a STING, IRF3 or IRF7 immunepotentiator mRNA construct.

FIGS. 9A-9B are graphs showing the percentage of CD8b⁺ cells among liveCD45⁺ cells in spleen or PBMCs (FIG. 9A) or the percentage of CD62L^(lo)cells among CD8b⁺ cell in spleen or PBMCs (FIG. 9B) from mice immunizedwith MC38 neo-antigen vaccine construct (ADRvax) coformulated witheither a STING, IRF3 or IRF7 immune potentiator mRNA construct.

FIG. 10 depicts NRAS and KRAS mutation frequency in colorectal cancer asidentified using cBioPortal.

FIGS. 11A-11B are graphs showing intracellular staining (ICS) of CD8⁺splenocytes from mice immunized with HPV E6/E7 vaccine constructscoformulated with either a STING, IRF3 or IRF7 immune potentiator mRNAconstruct on day 21 post first immunization. FIG. 11A shows E7-specificresponses for IFN-γ ICS. FIG. 11B shows E7-specific responses for TNF-αICS.

FIGS. 12A-12B are graphs showing intracellular staining (ICS) of CD8⁺splenocytes from mice immunized with HPV E6/E7 vaccine constructscoformulated with either a STING, IRF3 or IRF7 immune potentiator mRNAconstruct. FIG. 12A shows E6-specific responses for IFN-γ ICS. FIG. 12Bshows E6-specific responses for TNF-α ICS.

FIGS. 13A-13B are graphs showing E7-specific responses for IFN-γintracellular staining (ICS) of day 21 (FIG. 13A) or day 53 (FIG. 13B)CD8⁺ splenocytes from mice immunized with HPV E6/E7 vaccine constructscoformulated with either a STING, IRF3 or IRF7 immune potentiator mRNAconstruct.

FIGS. 14A-14B are graphs showing the percentage of CD8b⁺ cells among thelive CD45⁺ cells for day 21 (FIG. 14A) or day 53 (FIG. 14B) spleen cellsfrom mice immunized with HPV E6/E7 vaccine constructs coformulated witheither a STING, IRF3 or IRF7 immune potentiator mRNA construct.

FIGS. 15A-15B are graphs showing E7-MHC1-tetramer staining of day 21(FIG. 15A) or day 53 (FIG. 15B) CD8b⁺ splenocytes from mice immunizedwith HPV E6/E7 vaccine constructs coformulated with either a STING, IRF3or IRF7 immune potentiator mRNA construct.

FIGS. 16A-16D are graphs showing that the majority of E7-tetramer⁺ CD8⁺cells have an “effector memory” CD62L^(lo) phenotype, with comparison ofday 21 versus day 53 E7-tetramer⁺ CD8 cells demonstrating that this“effector-memory” CD62L^(lo) phenotype was maintained throughout thestudy. FIGS. 16A (d21) and 16B (d53) show increased % of CD8 witheffector memory ‘CD62Llo phenotype. FIGS. 16C and 16D show increased %of E7-tetramer+ CD8 are CD62Llo.

FIGS. 17A-17C are graphs showing tumor volume from mice vaccinatedprophylactically as indicated with HPV E6/E7 construct together with aSTING immune potentiator mRNA construct (alone or in combination withanti-CTLA-4 or anti-PD1 treatment), either prior to or at the time ofchallenge with a TC1 tumor that expresses HPV E7, showing inhibition oftumor growth by the HPV E6/E7+STING treatment. Certain mice were treatedon days −14 and −7 with soluble E6/E7+STING (FIG. 17A) or withintracellular E6/E7+STING (FIG. 17B), with tumor challenge on day 1.Other mice were treated on days 1 and 8 with soluble E6/E7+STING (FIG.17C), with tumor challenge on day 1.

FIGS. 18A-18I are graphs showing tumor volume from mice vaccinatedtherapeutically as indicated with HPV E6/E7 construct together with aSTING immune potentiator mRNA construct (FIG. 18A), alone or incombination with anti-CTLA-4 (FIG. 18B) or anti-PD1 treatment (FIG.18C), after challenge with a TC1 tumor that expresses HPV E7, showinginhibition of tumor growth by the HPV E6/E7+STING treatment. FIGS.18D-18I show control treatments.

FIG. 19 is a graph showing intracellular staining (ICS) of CD8⁺splenocytes for IFN-γ from mice immunized with an ADR vaccine constructcoformulated with a STING immune potentiator at the indicated Ag:STINGratios on day 21 post first immunization. CD8+ cells were restimulatedwith either the mutant ADR antigen composition (comprising threepeptides) or the wild-type ADR composition (as a control).

FIG. 20 is a graph showing intracellular staining (ICS) of CD8⁺splenocytes for TNF-α from mice immunized with an ADR vaccine constructcoformulated with a STING immune potentiator at the indicated Ag:STINGratios on day 21 post first immunization. CD8+ cells were restimulatedwith either the mutant ADR antigen composition (comprising threepeptides) or the wild-type ADR composition (as a control).

FIGS. 21A-21C are graphs showing intracellular staining (ICS) of CD8⁺splenocytes for IFN-γ from mice immunized with an ADR vaccine constructcoformulated with a STING immune potentiator at the indicated Ag:STINGratios on day 21 post first immunization. CD8+ cells were restimulatedwith either a mutant or wild-type (as a control) peptide containedwithin the ADR antigen composition. FIG. 21A shows responses to theAdpk1 peptide within the ADR composition. FIG. 21B shows the response tothe Reps1 peptide within the ADR composition. FIG. 21C shows theresponse to the Dpagt1 peptide within the ADR composition.

FIG. 22 is a graph showing antigen-specific T cell responses to MHCclass I epitopes within the CA-132 vaccine, as measured by ELISpotanalysis for IFN-γ, from mice treated with a coformulation of CA-132 andSTING immune potentiator, at the indicated different Ag:STING ratios.

FIGS. 23A-23B show results for Ag:STING ratio studies from miceimmunized with HPV E6/E7 vaccine construct coformulated with a STINGimmune potentiator mRNA construct. FIG. 23A shows intracellular staining(ICS) of CD8+ splenocytes for IFN-γ from mice immunized at the indicatedAg:STING ratios on day 21 post immunization. FIG. 23B shows H2-Kb/E7peptide-tetramer staining of day 21 CD8+ splenocytes from mice immunizedat the indicated Ag:STING ratios.

FIGS. 24A-24C are bar graphs showing TNFα intracellular staining (ICS)results for CD8+ T cells from cynomolgus monkeys vaccinated with HPVvaccine+STING constructs, followed by ex vivo stimulation with eitherHPV16 E6 peptide pool (FIG. 24A), HPV16 E7 peptide pool (FIG. 24B) ormedium (negative control) (FIG. 24C).

FIGS. 25A-25C are bar graphs showing IL-2 intracellular staining (ICS)results for CD8+ T cells from cynomolgus monkeys vaccinated with HPVvaccine+STING constructs, followed by ex vivo stimulation with eitherHPV16 E6 peptide pool (FIG. 25A), HPV16 E7 peptide pool (FIG. 25B) ormedium (negative control) (FIG. 25C).

FIG. 26 is a graph showing ELISA results for anti-E6 IgG in serum fromcynomolgus monkeys vaccinated/immunized with HPV vaccine+STINGconstructs.

FIG. 27 is a graph showing ELISA results for anti-E7 IgG in serum fromcynomolgus monkeys vaccinated/immunized with HPV vaccine+STINGconstructs.

FIG. 28 is a graph showing ELISA results for anti-E6 IgG in a two-folddilution series of day 25 serum from cynomolgus monkeys treated with HPVvaccine+STING construct at a 1:10 STING:Ag ratio.

FIGS. 29A-29B are graphs showing calculated titer values of ELISAresults for anti-E6 IgG (FIG. 29A) or anti-E7 IgG (FIG. 29B) in day 25serum from cynomolgus monkeys treated with HPV vaccine+STING constructat the indicated STING:Ag ratios.

FIG. 30 is a graph showing the intracellular staining (ICS) results forCD8+ splenocytes for IFNγ from mice immunized with mutant KRASvaccine+STING construct followed by ex vivo stimulation with KRAS-G12Vpeptide.

FIG. 31 is a graph showing the intracellular staining (ICS) results forCD8+ splenocytes for IFNγ from mice immunized with mutant KRASvaccine+STING construct followed by ex vivo stimulation with KRAS-G12Dpeptide.

FIG. 32 is a graph showing the intracellular staining (ICS) results orCD8+ splenocytes for IFNγ from mice immunized with mutant KRASvaccine+STING construct followed by ex vivo co-culture with Cos7 cellsvirally transduced with HLA*A11 allele and pulsed with KRAS-G12V.

FIG. 33 is a graph showing the intracellular staining (ICS) results orCD8+ splenocytes for IFN-g from mice immunized with mutant KRASvaccine+STING construct followed by ex vivo co-culture with Cos7 cellsvirally transduced with HLA*A11 allele and pulsed with KRAS-G12D.

FIG. 34 is a graph showing the intracellular staining (ICS) results orCD8+ splenocytes for IFN-g from mice immunized with an A11 viral epitopeconcatemer+STING construct followed by ex vivo stimulation withindividual viral epitopes.

DETAILED DESCRIPTION

Provided herein are immunomodulatory therapeutic compositions, includingmRNA compositions and/or lipid nanoparticles comprising the same,comprising one or more RNAs (e.g., messenger RNAs (mRNAs)) that cansafely direct the body's cellular machinery to produce a cancer proteinor fragment thereof of interest, e.g., an activating oncogene mutationpeptide. In some embodiments, the RNA is a modified RNA. Theimmunomodulatory therapeutic compositions and lipid nanoparticles of thepresent disclosure may be used to induce a balanced immune responseagainst cancers, comprising both cellular and humoral immunity, withoutrisking the possibility of insertional mutagenesis, for example.

Accordingly, in some aspects, the disclosure provides animmunomodulatory therapeutic composition, including a lipid-basedcomposition such as a lipid nanoparticles, comprising: one or more mRNAeach having an open reading frame encoding an activating oncogenemutation peptide, and optionally one or more mRNA each having an openreading frame encoding a polypeptide that enhances an immune response tothe activating oncogene mutation peptide in a subject, wherein theimmune response comprises a cellular or humoral immune.

In one aspect, the disclosure provides an immunomodulatory therapeuticcomposition comprising four different activating oncogene mutationpeptides (e.g., KRAS G12D, G12C, G12V and G13D), which is capable oftreating patients having any one of colorectal cancer, pancreacticcarcinoma, and non-small cell lung carcinoma. The ability to target tofour different mutations and three different cancers is a significantadvantage of the compositions and methods provided herein.

An mRNA encoding a polypeptide that enhances an immune response to theactivating oncogene mutation peptide in a subject is also referred toherein as “an immune potentiator mRNA” or “mRNA encoding an immunepotentiator” or simply “immune potentiator.” An enhanced immune responsecan be a cellular response, a humoral response or both. As used herein,a “cellular” immune response is intended to encompass immune responsesthat involve or are mediated by T cells, whereas a “humoral” immuneresponse is intended to encompass immune responses that involve or aremediated by B cells. An mRNA encoding an immune potentiator may enhancean immune response by, for example,

(i) stimulating Type I interferon pathway signaling;

(ii) stimulating NFkB pathway signaling;

(iii) stimulating an inflammatory response;

(iv) stimulating cytokine production; or

(v) stimulating dendritic cell development, activity or mobilization;and

(vi) a combination of any of (i)-(v).

As used herein, “stimulating Type I interferon pathway signaling” isintended to encompass activating one or more components of the Type Iinterferon signaling pathway (e.g., modifying phosphorylation,dimerization or the like of such components to thereby activate thepathway), stimulating transcription from an interferon-sensitiveresponse element (ISRE) and/or stimulating production or secretion ofType I interferon (e.g., IFN-α, IFN-β, IFN-ε, IFN-κ and/or IFN-ω). Asused herein, “stimulating NFkB pathway signaling” is intended toencompass activating one or more components of the NFkB signalingpathway (e.g., modifying phosphorylation, dimerization or the like ofsuch components to thereby activate the pathway), stimulatingtranscription from an NFkB site and/or stimulating production of a geneproduct whose expression is regulated by NFkB. As used herein,“stimulating an inflammatory response” is intended to encompassstimulating the production of inflammatory cytokines (including but notlimited to Type I interferons, IL-6 and/or TNFα). As used herein,“stimulating dendritic cell development, activity or mobilization” isintended to encompass directly or indirectly stimulating dendritic cellmaturation, proliferation and/or functional activity.

The present disclosure provides compositions, including mRNAcompositions and/or lipid nanoparticles comprising the same, whichinclude one or more mRNA constructs encoding a polypeptide that enhancesimmune responses to an activating oncogene mutation peptide (alsoreferred to herein as “an antigen of interest”), referred to herein asimmune potentiator mRNA or immune potentiator mRNAs, includingchemically modified mRNAs (mmRNAs). The immune potentiator mRNAs of thedisclosure enhance immune responses by, for example, activating Type Iinterferon pathway signaling such that antigen-specific responses to anantigen of interest (i.e., activating oncogene mutation peptide(s)) arestimulated.

The immune potentiator mRNAs of the disclosure enhance immune responsesto an exogenous antigen that is administered to the subject with theimmune potentiator mRNA (e.g., an mRNA construct encoding activatingoncogene mutation peptide(s) that is coformulated and coadministeredwith the immune potentiator mRNA or an mRNA construct encodingactivating oncogene mutation peptide(s) that is formulated andadministered separately from the immune potentiator mRNA).Administration of an immune potentiator mRNA enhances an immune responsein a subject by stimulating, for example, cytokine production, T cellsresponses (e.g., antigen-specific CD8⁺ or CD4⁺ T cell responses) or Bcell responses (e.g., antigen-specific antibody production) in thesubject.

In other aspects, the disclosure provides compositions, including mRNAcompositions and lipid nanoparticles, comprising one or more mRNAconstructs (e.g., one or more mmRNA constructs), wherein the one or moremRNA constructs encode an activating oncogene mutation peptide(s) and,in the same or a separate mRNA construct, encode a polypeptide thatenhances an immune response to the antigen of interest. In some aspects,the disclosure provides nanoparticles, e.g., lipid nanoparticles, whichinclude an immune potentiator mRNA that enhances an immune response,alone or in combination with mRNAs that encode activating oncogenemutation peptide(s). The disclosure also provides pharmaceuticalcompositions comprising any of the mRNAs as described herein ornanoparticles, e.g., lipid nanoparticles comprising any of the mRNAs asdescribed herein.

In other aspects, the disclosure provides methods for enhancing animmune response to an activating oncogene mutation peptide(s) byadministering to a subject one or more mRNAs encoding activatingoncogene mutation peptide(s) and a mRNA encoding a polypeptide thatenhances an immune response to the peptide(s) of interest, or lipidnanoparticle thereof, or pharmaceutical composition thereof, such thatan immune response to the activating oncogene mutation peptide(s) isenhanced in the subject. The methods of enhancing an immune response canbe used, for example, to stimulate an immunogenic response to a tumor ina subject.

Cancer Antigens of Interest

The immune potentiators mRNAs of the disclosure are useful incombination with any type of antigen for which enhancement of an immuneresponse is desired, including with mRNA sequences encoding at least oneantigen of interest (on either the same or a separate mRNA construct) toenhance immune responses against the antigen of interest, such as atumor antigen. Thus, the immune potentiator mRNAs of the disclosureenhance, for example, mRNA vaccine responses, thereby acting as geneticadjuvants.

Activating Oncogene Mutation Peptides

In one embodiment, the antigen(s) of interest is a tumor antigen. In oneembodiment, the tumor antigen comprises a tumor neoepitope, e.g., mutantpeptide from a tumor antigen. In one embodiment, the tumor antigen is aRas antigen. A comprehensive survey of Ras mutations in cancer has beendescribed in the art (Prior, I. A. et al. (2012) Cancer Res.72:2457-2467). Accordingly, a Ras amino acid sequence comprising atleast one mutation associated with cancer can be used as an antigen ofinterest. In one embodiment, the tumor antigen is a mutant KRAS antigen.Mutant KRAS antigens have been implicated in acquired resistance tocertain therapeutic agents (see e.g., Misale, S. et al. (2012) Nature486:532-536; Diaz, L. A. et al. (2012) Nature 486:537-540).

Although attempts have been made to produce functional immunomodulatorytherapeutic compositions, including mRNA compositions, the therapeuticefficacy of these RNA compositions has not yet been fully established.Quite surprisingly, the inventors have discovered a class offormulations for delivering mRNA immunomodulatory therapeuticcompositions that results in significantly enhanced, and in manyrespects synergistic, immune responses including enhanced T cellresponses. KRAS is the most frequently mutated oncogene in human cancer(˜15%). Such KRAS mutations are mostly conserved in a few “hotspots” andactivate the oncogene.

The immunomodulatory therapeutic compositions of the invention includeactivating oncogene mutation peptides, such as KRAS mutation peptides.Prior research has shown limited ability to raise T cells specific tothe oncogenic mutation. Much of this research was done in the context ofthe most common HLA allele (A2, which occurs in ˜50% of Caucasians).More recent work has explored the generation of specific T cells againstpoint mutations in the context of less common HLA alleles (A11, C8).These findings have significant implications for the treatment ofcancer. Oncogenic mutations are common in many cancers. The ability totarget these mutations and generate T cells that are sufficient to killtumors has broad applicability to cancer therapy. It is quite surprisingthat delivery of antigens using mRNA would have such a significantadvantage over the delivery of peptide vaccines. Thus the inventioninvolves, in some aspects, the surprising finding that activatingoncogenic mutation antigens delivered in vivo in the form of an mRNAsignificantly enhances the generation of T cell effector and memoryresponses.

HLA class I molecules are highly polymorphic trans-membraneglycoproteins composed of two polypeptide chains (heavy chain and lightchain). Human leukocyte antigen, the major histocompatibility complex inhumans, is specific to each individual and has hereditary features. Theclass I heavy chains are encoded by three genes: HLA-A, HLA-B and HLA-C.HLA class I molecules are important for establishing an immune responseby presenting endogenous antigens to T lymphocytes, which initiates achain of immune reactions that lead to tumor cell elimination bycytotoxic T cells. Altered levels of production of HLA class I antigensis a widespread phenomenon in malignancies and is accompanied bysignificant inhibition of anti-tumor T cell function. It represents oneof the main mechanisms used by cancer cells to evadeimmuno-surveillance. Down regulated levels of HLA class I antigens weredetected in 90% of NSCLC tumors (n=65). A reduction or loss of HLA wasdetected in 76% of pancreatic tumor samples (n=19). The expression ofHLA class I antigens in colon cancer was dramatically reduced orundetectable in 96% of tumor samples (n=25).

Mounting evidence suggests that two general strategies are utilized bytumor cells to escape immune surveillance: immunoselection (poorlyimmunogenic tumor cell variants) and immunosubversion (subversion of theimmune system). A correlation between changes in HLA class I antigensand the presence of KRAS codon 12 mutations was demonstrated, whichsuggests a possible inductive effect of KRAS codon 12 mutations on HLAclass I antigen regulation in cancer progression. Many frequent cancermutations are predicted to bind HLA Class I alleles with high-affinity(IC50<=50 nM)7 and may be suitable for prophylactic cancer vaccination.

The generation of cancer antigens that elicit a desired immune response(e.g. T-cell responses) against targeted polypeptide sequences inimmunomodulatory therapeutic development remains a challenging task. Theinvention involves technology to overcome hurdles associated with suchdevelopment. Through the use of the technology of the invention, it ispossible to elicit a desired immune response by selecting appropriateactivating oncogene mutation peptides and formulating the mRNA encodingpeptides for effective delivery in vivo.

The immunomodulatory therapeutic compositions provide unique therapeuticalternatives to peptide based or DNA vaccines. When the mRNA containingimmunomodulatory therapeutic composition is delivered to a cell, themRNA will be translated into a polypeptide by the intracellularmachinery which can then process the polypeptide into sensitivefragments capable of being presented on MDC and stimulating an immuneresponse against the tumor.

The immunomodulatory therapeutic compositions described herein includeat least one ribonucleic acid (RNA) polynucleotide having an openreading frame encoding at least one cancer antigenic polypeptide or animmunogenic fragment thereof (e.g., an immunogenic fragment capable ofinducing an immune response to cancer). The antigenic peptide includesan activating oncogenic mutation. In some preferred embodiments thecomposition is multiple epitopes of a mixture of activating oncogenicmutations. Many activating oncogenic mutations are known in the art.

When oncogenes are activated they can inhibit programmed cell deathand/or cause abnormal cellular proliferation. Such oncogene activationcan lead to cancer. The KRAS gene (Ki-ras2 Kirsten rat sarcoma viraloncogene homolog) is an oncogene that encodes a small GTPase transductorprotein. KRAS relays external signals to the cell nucleus andcontributes to regulation of cell division. Activating mutations in theKRAS gene impair the ability of the KRAS protein to switch betweenactive and inactive states. KRAS activation leads to cell transformationand increased resistance to chemotherapy and biological therapiestargeting epidermal growth factor receptors. (Jancik, Sylwia et al.Clinical Relevance of KRAS in Human Cancers, Journal of Biomedicine andBiotechnology, Volume 2010 Article ID 150960 (2010)). Human KRAS aminoacid sequence is provided below (UniProtKB P01116). KRAS mutations arecommon in many cancers, and G12 is the site of most common KRASmutations.

>sp|P01116|1-186 (SEQ ID NO: 166)MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQRVEDAFYTLVREIRQYRLKKISKEEKTPGCVKIKKC

Mutant N-RAS proteins are highly prevalent in certain types of cancersand are useful as cancer vaccines. For instance, 29% of CutaneousMelanoma involves a RAS mutation, of which 94% are of N-RAS origin. Thisrepresents about 2,500 new US cases/year of the most aggressive form ofmelanoma accounting for the majority of melanoma deaths. (Channing Der,Are A11 RAS Proteins Created Equal in Cancer?, Sep. 22, 2014,cancer.gov). There are 30,280 news cases of multiple myeloma annually,of which 26% are NRAS*. This represents ˜6,100 new NRAS* cases per year.Thus, the N-Ras vaccines described herein are useful in some embodimentsin the treatment of melanoma and multiple myeloma as well as othermalignancies that harbor NRAS mutations.

Accordingly, in some aspects, the present invention provides mRNAencoding peptide sequences resulting from certain activating mutationsin one or more oncogenes, not limited to missense SNVs and oftenresulting in alternative splicing, for use as targets for therapeuticvaccination. In some embodiments, the activating oncogene mutation is aKRAS mutation. In some embodiments, the KRAS mutation is a G12 mutation.In some embodiments, the G12 KRAS mutation is selected from a G12D,G12V, G12S, G12C, G12A, and a G12R KRAS mutation, e.g., the G12 KRASmutation is selected from a G12D, G12V, and a G12S KRAS mutation. Insome embodiments, the G12 KRAS mutation is selected from a G12D, G12V,and a G12C KRAS mutation. In other embodiments, the KRAS mutation is aG13 mutation, e.g., the G13 KRAS mutation is a G13D KRAS mutation. Insome embodiments, the activating oncogene mutation is a H-RAS or N-RASmutation.

In one embodiment, one or more mRNAs encode a mutant KRAS peptide(s)comprising an amino acid sequence having one or more mutations selectedfrom G12D, G12V, G13D and G12C, and combinations thereof. Non-limitingexamples of mutant KRAS antigens include those comprising one or more ofthe amino acid sequences shown in SEQ ID NOs: 36-41 and 72, 125.

CD8+ T cells specific for the G12D or G12V mutations can be restrictedby HLA-A*02:01, A*03:01; -A*11:01, -B*35:01, -Cw*08:02, and potentiallyothers. Accordingly, in some embodiments, a KRAS mutation is selectedfor inclusion in an immunomodulatory therapeutic composition for asubject having T cells that are restricted by HLA-A*02:01, A*03:01;-A*11:01, -B*35:01, or -Cw*08:02. In some embodiments, the subject has Tcells that are HLA-A*02:01 restricted.

In one embodiment, the mutant KRAS antigen is one or more mutant KRAS15-mer peptides comprising a mutation selected from G12D, G12V, G13D andG12C, non-limiting examples of which are shown in SEQ ID NO: 36-38 and125.

In another embodiment, the mutant KRAS antigen is one or more mutantKRAS 25-mer peptides comprising a mutation selected from G12D, G12V,G13D and G12C, non-limiting examples of which are shown in SEQ ID NO:39-41 and 72.

In another embodiment, the mutant KRAS antigen is one or more mutantKRAS 3×15mer peptides (3 copies of the 15-mer peptide) comprising amutation selected from G12D, G12V, G13D and G12C, non-limiting examplesof which are shown in SEQ ID NO: 42-44 and 183.

In another embodiment, the mutant KRAS antigen is one or more mutantKRAS 3×25mer peptides (three copies of the 25-mer peptide) comprising amutation selected from G12D, G12V, G13D and G12C, non-limiting examplesof which are shown in SEQ ID NO: 45-47 and 73.

In another embodiment, the mutant KRAS antigen is a 100-mer concatemerpeptide of the 25-mer peptides containing the G12D, G12V, G13D and G12Cmutations (i.e., a 100-mer concatemer of SEQ ID NOs: 39, 40, 41 and 72).Accordingly, in one embodiment, the mutant KRAS antigen comprises anmRNA construct encoding SEQ ID NOs: 39, 40, 41 and 72. Non-limitingexamples of nucleotide sequences encoding a concatemer of peptidescontaining G12D, G12V, G13D and G12C mutations include SEQ ID NO: 138,SEQ ID NO: 167 and SEQ ID NO: 169. Further description of mutant KRASantigens, amino acid sequences thereof, and mRNA sequences encodingtherefor, are disclosed in U.S. Application Ser. No. 62/453,465, theentire contents of which is expressly incorporated herein by reference.

Some embodiments of the present disclosure provide immunomodulatorytherapeutic compositions that include an mRNA having an open readingframe encoding a concatemer of two or more activating oncogene mutationpeptides. In some embodiments, at least two of the peptide epitopes areseparated from one another by a single Glycine. In some embodiments, theconcatemer comprises 3-10 activating oncogene mutation peptides. In somesuch embodiments, all of the peptide epitopes are separated from oneanother by a single Glycine. In other embodiments, at least two of thepeptide epitopes are linked directly to one another without a linker.

In one embodiment, a tumor antigen is encoded by an mRNA construct thatalso comprises an immune potentiator (i.e., also encodes a polypeptidethat enhances an immune response against the tumor antigen).Non-limiting examples of such constructs include the KRAS-STINGconstructs encoding one of the amino acid sequences shown in SEQ ID NOs:48-71. Non-limiting examples of nucleotide sequences encoding theKRAS-STING constructs are shown in SEQ ID NOs: 160-163 and 221-224.

The disclosure provides an immunomodulatory therapeutic composition,comprising: an mRNA having an open reading frame encoding a concatemerof two or more activating oncogene mutation peptides, wherein theconcatemer comprises KRAS activating oncogene mutation peptides G12D,G12V, G12C, and G13D; and one or more mRNA each having an open readingframe encoding a polypeptide that enhances an immune response to theKRAS activating oncogene mutation peptides in a subject, such as a STINGimmune potentiator mRNA. Such an immunomodulatory composition targetssomatic point mutations of KRAS, which constitute not only exquisitelyspecific tumor neoantigens but also significant oncogenic drivermutations in various malignancies. Unlike many neoantigens, which arelargely passenger mutations, maintenance of KRAS mutant expression isimportant to cancer cells' survival as it helps drive aberrant cellproliferation and is likely to be a truncal event (an early event andtherefore present in many tumor cells).

In order to model KRAS mutant antigens in preclinical studies describedherein examining the immune potentiating capacity of STING, two modelantigens were selected: (1) HPV E6 and E7 and (2) the ADR concatemer ofthree point mutations from three genes found in the murine cell lineMC38. These antigens are appropriate models of the KRAS mutant antigensfor a number of reasons. For example, HPV E6 and E7 are viral oncogenicproteins whose expression is vital for the transformed phenotype, likemutant KRAS. Accordingly, HPV E6 and E7 are suitable model antigensbecause, similar to mutant KRAS, they are oncogenic drivers. The threeADR mutant epitopes, in contrast, are stereotypical neoantigens in thatthey are most likely passenger mutations. However, ADR more effectivelymodels other properties of KRAS antigens encoded by our vaccine in that:(1) each antigen contains a single missense mutation relative to itswild-type counterpart which is likely to be more challenging torecognize as “non-self” by the immune system than a viral antigen and(2) they are concatemerized.

The immunomodulatory therapeutic compositions of the disclosure mayinclude one or more cancer antigens. In some embodiments theimmunomodulatory therapeutic composition is composed of 2 or more, 3 ormore, 4 or more, 5 or more 6 or more 7 or more, 8 or more, 9 or moreantigens, e.g., activating oncogene mutation peptides. In otherembodiments the immunomodulatory therapeutic composition is composed of1000 or less, 900 or less, 500 or less, 100 or less, 75 or less, 50 orless, 40 or less, 30 or less, 20 or less or 100 or less cancer antigens,e.g., activating oncogene mutation peptides. In yet other embodimentsthe immunomodulatory therapeutic composition has 3-10, 3-100, 5-100,10-100, 15-100, 20-100, 25-100, 30-100, 35-100, 40-100, 45-100, 50-100,55-100, 60-100, 65-100, 70-100, 75-100, 80-100, 90-100, 5-50, 10-50,15-50, 20-50, 25-50, 30-50, 35-50, 40-50, 45-50, 100-150, 100-200,100-300, 100-400, 100-500, 50-500, 50-800, 50-1,000, or 100-1,000 cancerantigens, e.g., activating oncogene mutation peptides.

An epitope, also known as an antigenic determinant, as used herein is aportion of an antigen that is recognized by the immune system in theappropriate context, specifically by antibodies, B cells, or T cells.Epitopes include B cell epitopes and T cell epitopes. B-cell epitopesare peptide sequences which are required for recognition by specificantibody producing B-cells. B cell epitopes refer to a specific regionof the antigen that is recognized by an antibody. The portion of anantibody that binds to the epitope is called a paratope. An epitope maybe a conformational epitope or a linear epitope, based on the structureand interaction with the paratope. A linear, or continuous, epitope isdefined by the primary amino acid sequence of a particular region of aprotein. The sequences that interact with the antibody are situated nextto each other sequentially on the protein, and the epitope can usuallybe mimicked by a single peptide. Conformational epitopes are epitopesthat are defined by the conformational structure of the native protein.These epitopes may be continuous or discontinuous, i.e. components ofthe epitope can be situated on disparate parts of the protein, which arebrought close to each other in the folded native protein structure.

T-cell epitopes are peptide sequences which, in association withproteins on APC, are required for recognition by specific T-cells. Tcell epitopes are processed intracellularly and presented on the surfaceof APCs, where they are bound to MHC molecules including MHC class IIand MHC class I. The peptide epitope may be any length that isreasonable for an epitope. In some embodiments the peptide epitope is9-30 amino acids. In other embodiments the length is 9-22, 9-29, 9-28,9-27, 9-26, 9-25, 9-24, 9-23, 9-21, 9-20, 9-19, 9-18, 10-22, 10-21,10-20, 11-22, 22-21, 11-20, 12-22, 12-21, 12-20, 13-22, 13-21, 13-20,14-19, 15-18, or 16-17 amino acids.

In some embodiments the immunomodulatory therapeutic composition mayinclude a recall antigen, also sometimes referred to as a memoryantigen. A recall antigen is an antigen that has previously beenencountered by an individual and for which there are pre-existent memorylymphocytes. In some embodiments the recall antigen may be an infectiousdisease antigen that the individual has likely encountered such as aninfluenza antigen. The recall antigen helps promote a more robust immuneresponse.

The therapeutic mRNA can be delivered alone or in combination with othercancer therapeutics such as checkpoint inhibitors to provide asignificantly enhanced immune response against tumors. The checkpointinhibitors can enhance the effects of the mRNA encoding activatingoncogenic peptides by eliminating some of the obstacles to promoting animmune response, thus allowing the activated T cells to efficientlypromote an immune response against the tumor.

The mRNA may be delivered to the subject in the form of carrier such asa lipid nanoparticle (LNP). A number of LNPs are known in the art. Forinstance some LNPs such as those which have been used previously todeliver siRNA various in animal models as well as in humans have beenobserved to cause an undesirable inflammatory response associated with atransient IgM response, typically leading to a reduction in antigenproduction and a compromised immune response. In contrast to thefindings observed with siRNA, lipid nanoparticle-mRNA immunomodulatorytherapeutic compositions are provided herein that generate T cellresponses sufficient for therapeutic methods rather than promotingtransient IgM responses. The LNPs described herein are not liposomes. Aliposome as used herein is a lipid based structure having a simple lipidbilayer shell with a nucleic acid payload in the core.

An mRNA construct encoding an antigen(s) of interest typicallycomprises, in addition to the antigen-encoding sequences, otherstructural properties as described herein for mRNA constructs (e.g.,modified nucleobases, 5′ cap, 5′ UTR, 3′ UTR, miR binding site(s), polyAtail, as described herein). Suitable mRNA construct components are asdescribed herein.

Personalized Cancer Antigens—Neoepitopes

The cancer antigens can be personalized cancer antigens. Personalizedimmunomodulatory therapeutic compositions, for instance, may include RNAencoding for one or more known cancer antigens specific for the tumor orcancer antigens specific for each subject, referred to as neoepitopes orsubject specific epitopes or antigens. A “subject specific cancerantigen” is an antigen that has been identified as being expressed in atumor of a particular patient. The subject specific cancer antigen mayor may not be typically present in tumor samples generally. Tumorassociated antigens that are not expressed or rarely expressed innon-cancerous cells, or whose expression in non-cancerous cells issufficiently reduced in comparison to that in cancerous cells and thatinduce an immune response induced upon vaccination, are referred to asneoepitopes. Neoepitopes, like tumor associated antigens, are completelyforeign to the body and thus would not produce an immune responseagainst healthy tissue or be masked by the protective components of theimmune system. In some embodiments personalized immunomodulatorytherapeutic compositions based on neoepitopes are desirable because suchvaccine formulations will maximize specificity against a patient'sspecific tumor. Mutation-derived neoepitopes can arise from pointmutations, non-synonymous mutations leading to different amino acids inthe protein; read-through mutations in which a stop codon is modified ordeleted, leading to translation of a longer protein with a noveltumor-specific sequence at the C-terminus; splice site mutations thatlead to the inclusion of an intron in the mature mRNA and thus a uniquetumor-specific protein sequence; chromosomal rearrangements that giverise to a chimeric protein with tumor-specific sequences at the junctionof 2 proteins (i.e., gene fusion); frameshift mutations or deletionsthat lead to a new open reading frame with a novel tumor-specificprotein sequence; and translocations. Thus, in some embodiments theimmunomodulatory therapeutic compositions include at least 1 cancerantigens including mutations selected from the group consisting offrame-shift mutations and recombinations or any of the other mutationsdescribed herein.

Methods for generating personalized immunomodulatory therapeuticcompositions generally involve identification of mutations, e.g., usingdeep nucleic acid or protein sequencing techniques, identification ofneoepitopes, e.g., using application of validated peptide-MHC bindingprediction algorithms or other analytical techniques to generate a setof candidate T cell epitopes that may bind to patient HLA alleles andare based on mutations present in tumors, optional demonstration ofantigen-specific T cells against selected neoepitopes or demonstrationthat a candidate neoepitope is bound to HLA proteins on the tumorsurface and development of the vaccine. The immunomodulatory therapeuticcompositions of the invention may include multiple copies of a singleneoepitope, multiple different neoepitopes based on a single type ofmutation, i.e. point mutation, multiple different neoepitopes based on avariety of mutation types, neoepitopes and other antigens, such as tumorassociated antigens or recall antigens.

Examples of techniques for identifying mutations include but are notlimited to dynamic allele-specific hybridization (DASH), microplatearray diagonal gel electrophoresis (MADGE), pyrosequencing,oligonucleotide-specific ligation, the TaqMan system as well as variousDNA “chip” technologies i.e. Affymetrix SNP chips, and methods based onthe generation of small signal molecules by invasive cleavage followedby mass spectrometry or immobilized padlock probes and rolling-circleamplification.

The deep nucleic acid or protein sequencing techniques are known in theart. Any type of sequence analysis method can be used. Nucleic acidsequencing may be performed on whole tumor genomes, tumor exomes(protein-encoding DNA), tumor transcriptomes, or exosomes. Real-timesingle molecule sequencing-by-synthesis technologies rely on thedetection of fluorescent nucleotides as they are incorporated into anascent strand of DNA that is complementary to the template beingsequenced. Other rapid high throughput sequencing methods also exist.Protein sequencing may be performed on tumor proteomes. Additionally,protein mass spectrometry may be used to identify or validate thepresence of mutated peptides bound to MHC proteins on tumor cells.Peptides can be acid-eluted from tumor cells or from HLA molecules thatare immunoprecipitated from tumor cells, and then identified using massspectrometry. The results of the sequencing may be compared with knowncontrol sets or with sequencing analysis performed on normal tissue ofthe patient.

Accordingly, the present invention relates to methods for identifyingand/or detecting neoepitopes of an antigen, such as T-cell epitopes.Specifically, the invention provides methods of identifying and/ordetecting tumor specific neoepitopes that are useful in inducing a tumorspecific immune response in a subject. Optionally, these neoepitopesbind to class I HLA proteins with a greater affinity than the wild-typepeptide and/or are capable of activating anti-tumor CD8 T-cells.Identical mutations in any particular gene are rarely found acrosstumors.

Proteins of MHC class I are present on the surface of almost all cellsof the body, including most tumor cells. The proteins of MHC class I areloaded with antigens that usually originate from endogenous proteins orfrom pathogens present inside cells, and are then presented to cytotoxicT-lymphocytes (CTLs). T-Cell receptors are capable of recognizing andbinding peptides complexed with the molecules of MHC class I. Eachcytotoxic T-lymphocyte expresses a unique T-cell receptor which iscapable of binding specific MHC/peptide complexes.

Using computer algorithms, it is possible to predict potentialneoepitopes such as T-cell epitopes, i.e. peptide sequences, which arebound by the MHC molecules of class I or class II in the form of apeptide-presenting complex and then, in this form, recognized by theT-cell receptors of T-lymphocytes. Examples of programs useful foridentifying peptides which will bind to MHC include for instance: LonzaEpibase, SYFPEITHI (Rammensee et al., Immunogenetics, 50 (1999),213-219) and HLA_BIND (Parker et al., J. Immunol., 152 (1994), 163-175).

Once putative neoepitopes are selected, they can be further tested usingin vitro and/or in vivo assays. Conventional in vitro lab assays, suchas Elispot assays may be used with an isolate from each patient, torefine the list of neoepitopes selected based on the algorithm'spredictions. Neoepitope vaccines, methods of use thereof and methods ofpreparing are all described in PCT/US2016/044918 which is incorporatedherein by reference in its entirety.

Endogenous Tumor Antigens

In another embodiment, the tumor antigen is an endogenous tumor antigen,such as a tumor antigen that is released upon destruction of tumor cellsin situ. It has been established in the art that natural mechanismsexist that results in cell death in vivo leading to release ofintracellular components such that an immune response may be stimulatedagainst the intracellular components. Such mechanisms are referred toherein as immunogenic cell death and include necroptosis and pyroptosis.Accordingly, in one embodiment, an immune potentiator mRNA construct ofthe disclosure is administered to a tumor-bearing subject underconditions in which endogenous immunogenic cell death is occurring suchthat one or more endogenous tumor antigens are released, to therebyenhance an immune response against the tumor antigens. In oneembodiment, the immune potentiator mRNA construct is administered to atumor-bearing subject together with a second mRNA construct encoding an“executioner mRNA construct”, which stimulates immunogenic cell death oftumor cells in the subject. Examples of executioner mRNA constructsinclude those encoding MLKL, RIPK3, RIPK1, DIABLO, FADD, GSDMD,caspase-4, caspase-5, caspase-11, Pyrin, NLRP3 and ASC/PYCARD.Executioner mRNA constructs, and their use in combination with an immunepotentiator mRNA construct, are described in further detail in U.S.Application Ser. No. 62/412,933, the entire contents of which isexpressly incorporated herein by reference.

Characteristics of Cancer Antigens

The activating oncogene mutation peptides selected for inclusion in theimmunomodulatory therapeutic composition typically will be high affinitybinding peptides. In some aspect the activating oncogene mutationpeptide binds an HLA protein with greater affinity than a wild-typepeptide. The activating oncogene mutation peptides has an IC50 of atleast less than 5000 nM, at least less than 500 nM, at least less than250 nM, at least less than 200 nM, at least less than 150 nM, at leastless than 100 nM, at least less than 50 nM or less in some embodiments.Typically, peptides with predicted IC50<50 nM, are generally consideredmedium to high affinity binding peptides and will be selected fortesting their affinity empirically using biochemical assays ofHLA-binding.

In some embodiments, subject specific activating oncogene mutationpeptides may be identified in a sample of a patient. For instance, thesample may be a tissue sample or a tumor sample. For instance, a sampleof one or more tumor cells may be examined for the presence of subjectspecific activating oncogene mutations. The tumor sample may be examinedusing whole genome, exome or transcriptome analysis in order to identifythe subject specific activating oncogene mutations.

Alternatively the subject specific activating oncogene mutation peptidesmay be identified in an exosome of the subject. When the activatingoncogene mutation peptides are identified in an exosome of the subject,such peptides are said to be representative of exosome peptides of thesubject.

Exosomes are small microvesicles shed by cells, typically having adiameter of approximately 30-100 nm. Exosomes are classically formedfrom the inward invagination and pinching off of the late endosomalmembrane, resulting in the formation of a multivesicular body (MVB)laden with small lipid bilayer vesicles, each of which contains a sampleof the parent cell's cytoplasm. Fusion of the MVB with the cell membraneresults in the release of these exosomes from the cell, and theirdelivery into the blood, urine, cerebrospinal fluid, or other bodilyfluids. Exosomes can be recovered from any of these biological fluidsfor further analysis.

Nucleic acids within exosomes have a role as biomarkers for tumorantigens. An advantage of analyzing exosomes in order to identifysubject specific cancer antigens, is that the method circumvents theneed for biopsies. This can be particularly advantageous when thepatient needs to have several rounds of therapy including identificationof cancer antigens, and vaccination.

A number of methods of isolating exosomes from a biological sample havebeen described in the art. For example, the following methods can beused: differential centrifugation, low speed centrifugation, anionexchange and/or gel permeation chromatography, sucrose density gradientsor organelle electrophoresis, magnetic activated cell sorting (MACS),nanomembrane ultrafiltration concentration, Percoll gradient isolationand using microfluidic devices. Exemplary methods are described in USPatent Publication No. 2014/0212871 for instance.

Immune Potentiator mRNAs

One aspect of the disclosure pertains to mRNAs that encode a polypeptidethat stimulates or enhances an immune response against one or moreantigens of interest (activating oncogene mutation peptide(s)). SuchmRNAs that enhance immune responses to an antigen(s) of interest arereferred to herein as immune potentiator mRNA constructs or immunepotentiator mRNAs, including chemically modified mRNAs (mmRNAs). In someaspects, the disclosure provides an mRNA encoding a polypeptide thatstimulates or enhances an immune response in a subject in need thereof(e.g., potentiates an immune response in the subject) by, for example,inducing adaptive immunity (e.g., by stimulating Type I interferonproduction), stimulating an inflammatory response, stimulating NFkBsignaling and/or stimulating dendritic cell (DC) development, activityor mobilization in the subject. In some aspects, administration of animmune potentiator mRNA to a subject in need thereof enhances cellularimmunity (e.g., T cell-mediated immunity), humoral immunity (e.g., Bcell-mediated immunity) or both cellular and humoral immunity in thesubject. In some aspects, administration of an immune potentiator mRNAstimulates cytokine production (e.g., inflammatory cytokine production),stimulates antigen-specific CD8⁺ effector cell responses, stimulatesantigen-specific CD4⁺ helper cell responses, increases the effectormemory CD62L^(lo) T cell population, stimulates B cell activity orstimulates antigen-specific antibody production, including combinationsof the foregoing responses.

In some aspects, administration of an immune potentiator mRNA stimulatescytokine production (e.g., inflammatory cytokine production) andstimulates antigen-specific CD8⁺ effector cell responses. In someaspects, administration of an immune potentiator mRNA stimulatescytokine production (e.g., inflammatory cytokine production), andstimulates antigen-specific CD4⁺ helper cell responses. In some aspects,administration of an immune potentiator mRNA stimulates cytokineproduction (e.g., inflammatory cytokine production), and increases theeffector memory CD62L^(lo) T cell population. In some aspects,administration of an immune potentiator mRNA stimulates cytokineproduction (e.g., inflammatory cytokine production), and stimulates Bcell activity or stimulates antigen-specific antibody production.

Immune Potentiators mRNAs that Stimulate Type I Interferon

In some aspects, the disclosure provides an immune potentiator mRNAencoding a polypeptide that stimulates or enhances an immune responseagainst an antigen of interest by simulating or enhancing Type Iinterferon pathway signaling, thereby stimulating or enhancing Type Iinterferon (IFN) production. It has been established that successfulinduction of anti-tumor or anti-microbial adaptive immunity requiresType I IFN signaling (see e.g., Fuertes, M. B. et al. (2013) TrendsImmunol. 34:67-73). The production of Type I IFNs (including IFN-α,IFN-β, IFN-ε, IFN-κ and IFN-ω) plays a role in clearance of microbialinfections, such as viral infections. It has also been appreciated thathost cell DNA (for example derived from damaged or dying cells) iscapable of inducing Type I interferon production and that the Type I IFNsignaling pathway plays a role in the development of adaptive anti-tumorimmunity. However, many pathogens and cancer cells have evolvedmechanisms to reduce or inhibit Type I interferon responses. Thus,activation (including stimulation and/or enhancement) of the Type I IFNsignaling pathway in a subject in need thereof, by providing an immunepotentiator mRNA of the disclosure to the subject, stimulates orenhances an immune response in the subject in a wide variety of clinicalsituations, including treatment of cancer and pathogenic infections, aswell as in potentiating vaccine responses to provide protectiveimmunity.

Type I interferons (IFNs) are pro-inflammatory cytokines that arerapidly produced in multiple different cell types, typically upon viralinfection, and known to have a wide variety of effects. The canonicalconsequences of type I IFN production in vivo is the activation ofantimicrobial cellular programs and the development of innate andadaptive immune responses. Type I IFN induces a cell-intrinsicantimicrobial state in infected and neighboring cells that limits thespread of infectious agents, particularly viral pathogens. Type I IFNalso modulates innate immune cell activation (e.g., maturation ofdendritic cells) to promote antigen presentation and nature killer cellfunctions. Type I IFN also promotes the development of high-affinityantigen-specific T and B cell responses and immunological memory(Ivashkiv and Donlin (2014) Nat Rev Immunol 14(1):36-49).

Type I IFN activates dendritic cells (DCs) and promotes their T cellstimulatory capacity through autocrine signaling (Montoya et al., (2002)Blood 99:3263-3271). Type I IFN exposure facilitates maturation of DCsvia increasing the expression of chemokine receptors and adhesionmolecules (e.g., to promote DC migration into draining lymph nodes),co-stimulatory molecules, and MHC class I and class II antigenpresentation. DCs that mature following type I IFN exposure caneffectively prime protective T cell responses (Wijesundara et al.,(2014) Front Immunol 29(412) and references therein).

Type I IFN can either promote or inhibit T cell activation,proliferation, differentiation and survival depending largely on thetiming of type I IFN signaling relative to T cell receptor signaling(Crouse et al., (2015) Nat Rev Immunol 15:231-242). Early studiesrevealed that MHC-I expression is upregulated in response to type I IFNin multiple cell types (Lindahl et al., (1976), J Infect Dis133(Suppl):A66-A68; Lindahl et al., (1976) Proc Natl Acad Sci USA17:1284-1287) which is a requirement for optimal T cell stimulation,differentiation, expansion and cytolytic activity. Type I IFN can exertpotent co-stimulatory effects on CD8 T cells, enhancing CD8 T cellproliferation and differentiation (Curtsinger et al., (2005) J Immunol174:4465-4469; Kolumam et al., (2005) J Exp Med 202:637-650).

Similar to effects on T cells, type I IFN signaling has both positiveand negative effects on B cell responses depending on the timing andcontext of exposure (Braun et al., (2002) Int Immunol 14(4):411-419; Linet al, (1998) 187(1):79-87). The survival and maturation of immature Bcells can be inhibited by type I IFN signaling. In contrast to immatureB cells, type I IFN exposure has been shown to promote B cellactivation, antibody production and isotype switch following viralinfection or following experimental immunization (Le Bon et al., (2006)J Immunol 176:4:2074-2078; Swanson et al., (2010) J Exp Med207:1485-1500).

A number of components involved in Type I IFN pathway signaling havebeen established, including STING, Interferon Regulatory Factors, suchas IRF1, IRF3, IRF5, IRF7, IRF8, and IRF9, TBK1, IKKi, MyD88 and TRAM.Additional components involved in Type I IFN pathway signaling includeTRAF3, TRAF6, IRAK-1, IRAK-4, TRIF, IPS-1, TLR-3, TLR-4, TLR-7, TLR-8,TLR-9, RIG-1, DAI and IFI16.

Accordingly, in one embodiment, an immune potentiator mRNA encodes anyof the foregoing components involved in Type I IFN pathway signaling.

Immune Potentiator mRNA Encoding STING

The present disclosure encompasses mRNA (including mmRNA) encodingSTING, including constitutively active forms of STING, as immunepotentiators. STING (STimulator of INterferon Genes; also known astransmembrane protein 173 (TMEM173), mediator of IRF3 activation (MITA),methionine-proline-tyrosine-serine (MPYS), and ER IFN stimulator (ERIS))is a 379 amino acid, endoplasmic reticulum (ER) resident transmembraneprotein that functions as a signaling molecule controlling thetranscription of immune response genes, including type I IFNs andpro-inflammatory cytokines (Ishikawa & Barber, (2008) Nature455:647-678; Ishikawa et al., (2009) Nature 461:788-792; Barber (2010)Nat Rev Immunol 15(12):760-770).

STING functions as a signaling adaptor linking the cytosolic detectionof DNA to the TBK1/IRF3/Type I IFN signaling axis. The signaling adaptorfunctions of STING are activated through the direct sensing of cyclicdinucleotides (CDNs). Examples of CDNs include cyclic di-GMP (guanosine5′-monophosphate), cyclic di-AMP (adenosine 5′-monophosphate) and cyclicGMP-AMP (cGAMP). Initially characterized as ubiquitous bacterialsecondary messengers, CDNs are now known to constitute a class ofpathogen-associated molecular pattern molecules (PAMPs) that activatethe TBK1/IRF3/type I IFN signaling axis via direct interaction withSTING. STING is capable of sensing aberrant DNA species and/or CDNs inthe cytosol of the cell, including CDNs derived from bacteria, and/orfrom the host protein cyclic GMP-AMP synthase (cGAS). The cGAS proteinis a DNA sensor that produces cGAMP in response to detection of DNA inthe cytosol (Burdette et al., (2011) Nature 478:515-518; Sun et al.,(2013) Science 339:786-791; Diner et al., (2013) Cell Rep 3:1355-1361;Ablasser et al., (2013) Nature 498:380-384).

Upon binding to a CDN, STING dimerizes and undergoes a conformationalchange that promotes formation of a complex with TANK-binding kinase 1(TBK1) (Ouyang et al., (2012) Immunity 36(6):1073-1086). This complextranslocates to the perinuclear Golgi, resulting in delivery of TBK1 toendolysosomal compartments where it phosphorylates IRF3 and NF-κBtranscription factors (Zhong et al., (2008) Immunity 29:538-550). Arecent study has shown that STING functions as a scaffold by binding toboth TBK1 and IRF3 to specifically promote the phosphorylation of IRF3by TBK1 (Tanaka & Chen, (2012) Sci Signal 5(214):ra20). Activation ofthe IRF3-, IRF7- and NF-κB-dependent signaling pathways induces theproduction of cytokines and other immune response-related proteins, suchas type I IFNs, which promote anti-pathogen and/or anti-tumor activity.

A number of studies have investigated the use of CDN agonists of STINGas potential vaccine adjuvants or immunomodulatory agents to elicithumoral and cellular immune responses (Dubensky et al., (2013) Ther AdvVaccines 1(4):131-143 and references therein). Initial studiesdemonstrated that administration of the CDN c-di-GMP attenuatedStaphylococcus aureus infection in vivo, reducing the number ofrecovered bacterial cells in a mouse infection model yet c-di-GMP had noobservable inhibitory or bactericidal effect on bacterial cells in vitrosuggesting the reduction in bacterial cells was due to an effect on thehost immune system (Karaolis et al., (2005) Antimicrob Agents Chemother49:1029-1038; Karaolis et al., (2007) Infect Immun 75:4942-4950). Recentstudies have shown that synthetic CDN derivative molecules formulatedwith granulocyte-macrophage colony-stimulating factor (GM-CSF)-producingcancer vaccines (termed STINGVAX) elicit enhanced in vivo antitumoreffects in therapeutic animal models of cancer as compared toimmunization with GM-CSF vaccine alone (Fu et al., (2015) Sci Transl Med7(283):283ra52), suggesting that CDN are potent vaccine adjuvants.

Mutant STING proteins resulting from polymorphisms mapped to the humanTMEM173 gene have been described exhibiting a gain-of function orconstitutively active phenotype. When expressed in vitro, mutant STINGalleles were shown to potently stimulate induction of type I IFN (Liu etal., (2014) N Engl J Med 371:507-518; Jeremiah et al., (2014) J ClinInvest 124:5516-5520; Dobbs et al., (2015) Cell Host Microbe18(2):157-168; Tang & Wang, (2015) PLoS ONE 10(3):e0120090; Melki etal., (2017) J Allergy Clin Immunol In Press; Konig et al., (2017) AnnRheum Dis 76(2):468-472; Burdette et al. (2011) Nature 478:515-518).

Provided herein are mRNAs (e.g., mmRNAs) encoding constitutively activeforms of STING, including mutant human STING isoforms for use as immunepotentiators as described herein. mmRNAs encoding constitutively activeforms of STING, including mutant human STING isoforms are set forth inthe Sequence Listing herein. The amino acid residue numbering for mutanthuman STING polypeptides used herein corresponds to that used for the379 amino acid residue wild type human STING (isoform 1) available inthe art as Genbank Accession Number NP_938023.

Accordingly, in one aspect, the disclosure provides a mRNA (e.g., mmRNA)encoding a mutant human STING protein having a mutation at amino acidresidue 155, in particular an amino acid substitution, such as a V155Mmutation. In one embodiment, the mRNA (e.g., mmRNAs) encodes an aminoacid sequence as set forth in SEQ ID NO:1. In one embodiment, the STINGV155M mutant is encoded by a nucleotide sequence shown in SEQ ID NO:139, SEQ ID NO: 168 or SEQ ID NO: 170. In one embodiment, the mRNA(e.g., mmRNAs) comprises a 3′ UTR sequence as shown in SEQ ID NO: 149,which includes an miR122 binding site.

In other aspects, the disclosure provides a mRNA (e.g., mmRNA) encodinga mutant human STING protein having a mutation at amino acid residue284, such as an amino acid substitution. Non-limiting examples ofresidue 284 substitutions include R284T, R284M and R284K. In certainembodiments, the mutant human STING protein has as a R284T mutation, forexample has the amino acid sequence set forth in SEQ ID NO: 2 or isencoded by an the nucleotide sequence shown in SEQ ID NO: 140 or 201. Incertain embodiments, the mutant human STING protein has a R284Mmutation, for example has the amino acid sequence as set forth in SEQ IDNO: 3 or is encoded by the nucleotide sequence shown in SEQ ID NO: 141or 202. In certain embodiments, the mutant human STING protein has aR284K mutation, for example has the amino acid sequence as set forth inSEQ ID NO: 4 or 164, or is encoded by the nucleotide sequence shown inSEQ ID NO: 142, 165, 203 or 225.

In other aspects, the disclosure provides a mRNA (e.g., mmRNA) encodinga mutant human STING protein having a mutation at amino acid residue154, such as an amino acid substitution, such as a N154S mutation. Incertain embodiments, the mutant human STING protein has a N154Smutation, for example has the amino acid sequence as set forth in SEQ IDNO: 5 or is encoded by the nucleotide sequence shown in SEQ ID NO: 143or 204.

In yet other aspects, the disclosure provides a mRNA (e.g., mmRNA)encoding a mutant human STING protein having a mutation at amino acidresidue 147, such as an amino acid substitution, such as a V147Lmutation. In certain embodiments, the mutant human STING protein havinga V147L mutation has the amino acid sequence as set forth in SEQ ID NO:6 or is encoded by the nucleotide sequence shown in SEQ ID NO: 144 or205.

In other aspects, the disclosure provides a mRNA (e.g., mmRNA) encodinga mutant human STING protein having a mutation at amino acid residue315, such as an amino acid substitution, such as a E315Q mutation. Incertain embodiments, the mutant human STING protein having a E315Qmutation has the amino acid sequence as set forth in SEQ ID NO: 7 or isencoded by the nucleotide sequence shown in SEQ ID NO: 145 or 206.

In other aspects, the disclosure provides a mRNA (e.g., mmRNA) encodinga mutant human STING protein having a mutation at amino acid residue375, such as an amino acid substitution, such as a R375A mutation. Incertain embodiments, the mutant human STING protein having a R375Amutation has the amino acid sequence as set forth in SEQ ID NO: 8 or isencoded by the nucleotide sequence shown in SEQ ID NO: 146 or 207.

In other aspects, the disclosure provides a mRNA (e.g., mmRNA) encodinga mutant human STING protein having a one or more or a combination oftwo, three, four or more of the foregoing mutations. Accordingly, in oneaspect the disclosure provides a mRNA (e.g., mmRNA) encoding a mutanthuman STING protein having one or more mutations selected from the groupconsisting of: V147L, N154S, V155M, R284T, R284M, R284K, E315Q andR375A, and combinations thereof. In other aspects, the disclosureprovides a mRNA (e.g., mmRNA) encoding a mutant human STING proteinhaving a combination of mutations selected from the group consisting of:V155M and R284T; V155M and R284M; V155M and R284K; V155M and V147L;V155M and N154S; V155M and E315Q; and V155M and R375A.

In other aspects, the disclosure provides a mRNA (e.g., mmRNA) encodinga mutant human STING protein having a V155M and one, two, three or moreof the following mutations: R284T; R284M; R284K; V147L; N154S; E315Q;and R375A. In other aspects, the disclosure provides a mRNA (e.g.,mmRNA) encoding a mutant human STING protein having V155M, V147L andN154S mutations. In other aspects, the disclosure provides a mRNA (e.g.,mmRNA) encoding a mutant human STING protein having V155M, V147L, N154Smutations, and, optionally, a mutation at amino acid 284. In yet otheraspects, the disclosure provides a mRNA (e.g., mmRNA) encoding a mutanthuman STING protein having V155M, V147L, N154S mutations, and a mutationat amino acid 284 selected from R284T, R284M and R284K. In otheraspects, the disclosure provides a mRNA (e.g., mmRNA) encoding a mutanthuman STING protein having V155M, V147L, N154S, and R284T mutations. Inother aspects, the disclosure provides a mRNA (e.g., mmRNA) encoding amutant human STING protein having V155M, V147L, N154S, and R284Mmutations. In other aspects, the disclosure provides a mRNA (e.g.,mmRNA) encoding a mutant human STING protein having V155M, V147L, N154S,and R284K mutations.

In other embodiments, the disclosure provides a mRNA (e.g., mmRNA)encoding a mutant human STING protein having a combination of mutationsat amino acid residue 147, 154, 155 and, optionally, 284, in particularamino acid substitutions, such as a V147L, N154S, V155M and, optionally,R284M. In certain embodiments, the mutant human STING protein has V147N,N154S and V155M mutations, such as the amino acid sequence as set forthin SEQ ID NO: 9 or encoded by the nucleotide sequence shown in SEQ IDNO: 147. In certain embodiments, the mutant human STING protein hasR284M, V147N, N154S and V155M mutations, such as the amino acid sequenceas set forth in SEQ ID NO: 10 or encoded by the nucleotide sequenceshown in SEQ ID NO: 148 or 209.

In another embodiment, the disclosure provides a mRNA (e.g., mmRNA)encoding a mutant human STING protein that is a constitutively activetruncated form of the full-length 379 amino acid wild type protein, suchas a constitutively active human STING polypeptide consisting of aminoacids 137-379.

Immune Potentiator mRNA Encoding Immune Regulatory Factor (IRF)

The present disclosure provides mRNA (including mmRNA) encodingInterferon Regulatory Factors, such as IRF1, IRF3, IRF5, IRF7, IRF8, andIRF9 as immune potentiators. The IRF transcription factor family isinvolved in the regulation of gene expression leading to the productionof type I interferons (IFNs) during innate immune responses. Nine humanIRFs have been identified to date (IRF-1-IRF-9), with each family membersharing extensive sequence homology within their N-terminal bindingdomains (DBDs) (Mamane et al., (1999) Gene 237:1-14; Taniguchi et al.,(2001) Annu Rev Immunol 19:623-655). Within the IRF family, IRF1, IRF3,IRF5, and IRF7 have been specifically implicated as positive regulatorsof type I IFN gene transcription (Honda et al., (2006) Immunity25(3):349-360). IRF1 was the first family member discovered to activatetype I IFN gene promoters (Miyamoto et al., (1988) Cell 54:903-913).Although studies show that IRF1 participates in type I IFN geneexpression, normal induction of type I IFN was observed invirus-infected IRF1−/− murine fibroblasts, suggesting dispensability(Matsuyama et al., (1993) Cell 75:83-97). IRF5 was also shown to bedispensable for type I IFN induction by viruses or TLR agonists (Takaokaet al., (2005) Nature 434:243-249).

Accordingly, in some aspects, the disclosure provides mRNA encodingconstitutively active forms of human IRF1, IRF3, IRF5, IRF7, IRF8, andIRF9 as immune potentiators. In some aspects, the disclosure providesmRNA encoding constitutively active forms of human IRF3 and/or IRF7.

During innate immune responses, IRF-3 plays a critical role in the earlyinduction of type I IFNs. The IRF3 transcription factor isconstitutively expressed and shuttles between the nucleus and cytoplasmof cells in latent form, with a predominantly cytosolic localizationprior to phosphorylation (Hiscott (2007) J Biol Chem282(21):15325-15329; Kumar et al., (2000) Mol Cell Biol20(11):4159-4168). Upon phosphorylation of serine residues at theC-terminus by TBK-1 (TANK binding kinase 1; also known as T2K and NAK)and/or IKKε (inducible IκB kinase; also known as IKKi), IRF3translocates from the cytoplasm into the nucleus (Fitzgerald et al.,(2003) Nat Immuno 4(5):491-496; Sharma et al., (2003) Science300:1148-1151; Hemmi et al., (2004) J Exp Med 199:1641-1650). Thetranscriptional activity of IRF3 is mediated by these phosphorylationand translocation events. A model for IRF3 activation proposes thatC-terminal phosphorylation induces a conformational change in IRF3 thatpromotes homo- and/or heterodimerization (e.g. with IRF7; see Honda etal., (2006) Immunity 25(3):346-360), nuclear localization, andassociation with the transcriptional co-activators CBP and/or p300 (Linet al., (1999) Mol Cell Biol 19(4):2465-2474). While inactive IRF3constitutively shuttles into and out of the nucleus, phosphorylated IRF3proteins remain associated with CBP and/or p300, are retained in thenucleus, and induce transcription of IFN and other genes (Kumar et al.,(2000) Mol Cell Biol 20(11):4159-4168).

In contrast to IRF3, IRF7 exhibits a low expression level in most cells,but is strongly induced by type I IFN-mediated signaling, supporting thenotion that IRF3 is primarily responsible for the early induction of IFNgenes and that IRF7 is involved in the late induction phase (Sato etal., (2000) Immunity 13(4):539-548). Ligand-binding to the type I IFNreceptor results in the activation of a heterotrimeric transcriptionalactivator, termed IFN-stimulated gene factor 3 (ISGF3), which consistsof IRF9, STAT1, and STAT2, and is responsible for the induction of theIRF7 gene (Marie et al., (1998) EMBO J 17(22):6660-6669). Like IRF3,IRF7 can partition between cytoplasm and nucleus after serinephosphorylation of its C-terminal region, allowing its dimerization andnuclear translocation. IRF7 forms a homodimer or a heterodimer withIRF3, and each of these different dimers differentially acts on the typeI IFN gene family members. IRF3 is more potent in activating the IFN-βgene than the IFN-α genes, whereas IRF7 efficiently activates both IFN-αand IFN-β genes (Marie et al., (1998) EMBO J 17(22):6660-6669).

Provided herein are mRNAs (e.g., mmRNAs) encoding constitutively activeforms of IRF3 and IRF7 including mutant human IRF3 and mutant human IRF7isoforms for use as immune potentiators as described herein. mRNAs(e.g., mmRNAs) encoding constitutively active forms of IRF3 and IRF7,including mutant human IRF3 and IRF7 isoforms are set forth in theSequence Listing herein. The amino acid residue numbering for mutanthuman IRF3 polypeptides used herein corresponds to that used for the 427amino acid residue wild type human IRF3 (isoform 1) available in the artas Genbank Accession Number NP_001562. The amino acid residue numberingfor mutant human IRF7 polypeptides used herein corresponds to that usedfor the 503 amino acid residue wild type human IRF7 (isoform a)available in the art as Genbank Accession Number NP_001563.

Accordingly, in some aspects, the disclosure provides a mRNA (e.g.,mmRNA) encoding a mutant human IRF3 protein that is constitutivelyactive, e.g., having a mutation at amino acid residue 396, such as anamino acid substitution, such as a S396D mutation, for example as setforth in the amino acid sequence of SEQ ID NO: 12 or encoded by thenucleotide sequence shown in SEQ ID NO: 151 or 212. In other aspects,the mRNA (e.g., mmRNA) construct encodes a constitutively active mouseIRF3 polypeptide comprising an S396D mutation, for example as set forthin the amino acid sequence of SEQ ID NO: 11 or encoded by the nucleotidesequence shown in SEQ ID NO: 150 or 211.

In other aspects, the disclosure provides a mRNA (e.g., mmRNA) encodinga mutant human IRF7 protein that is constitutively active. In oneaspect, the disclosure provides a mRNA (e.g., mmRNA) encoding aconstitutively active IR7 protein comprising one or more point mutations(amino acid substitutions compared to wild-type). In other aspects, thedisclosure provides a mRNA (e.g., mmRNA) encoding a constitutivelyactive IR7 protein comprising a truncated form of the protein (aminoacid deletions compared to wild-type). In yet other aspects, thedisclosure provides a mRNA (e.g., mmRNA) encoding a constitutivelyactive IR7 protein comprising a truncated form of the protein that alsoincludes one or more point mutations (a combination of amino aciddeletions and amino acid substitutions compared to wild-type).

The wild-type amino acid sequence of human IRF7 (isoform a) is set forthin SEQ ID NO: 13, encoded by the nucleotide sequence shown in SEQ ID NO:152 or 213. A series of constitutively active forms of human IRF7 wereprepared comprising point mutations, deletions, or both, as compared tothe wild-type sequence. In one aspect, the disclosure provides an immunepotentiator mRNA construct encoding a constitutively active IRF7polypeptide comprising one or more of the following mutations: S475D,S476D, S477D, S479D, L480D, S483D and S487D, and combinations thereof.In other aspects, the disclosure provides a mRNA (e.g., mmRNA) encodinga constitutively active IRF7 polypeptide comprising mutations S477D andS479D, as set forth in the amino acid sequence of SEQ ID NO: 14, encodedby the nucleotide sequence shown in SEQ ID NO: 153 or 214. In anotheraspect, the disclosure provides a mRNA (e.g., mmRNA) encoding aconstitutively active IRF7 polypeptide comprising mutations S475D, S477Dand L480D, as set forth in the amino acid sequence of SEQ ID NO: 15,encoded by the nucleotide sequence shown in SEQ ID NO: 154 or 215. Inother aspects, the disclosure provides a mRNA (e.g., mmRNAs) encoding aconstitutively active IRF7 polypeptide comprising mutations S475D,S476D, S477D, S479D, S483D and S487D, as set forth in the amino acidsequence of SEQ ID NO: 16, encoded by the nucleotide sequence shown inSEQ ID NO: 155 or 216. In another aspect, the disclosure provides a mRNA(e.g., mmRNA) encoding a constitutively active IRF7 polypeptidecomprising a deletion of amino acid residues 247-467 (i.e., comprisingamino acid residues 1-246 and 468-503), as set forth in the amino acidsequence of SEQ ID NO: 17, encoded by the nucleotide sequence shown inSEQ ID NO: 156 or 217. In yet other aspects, the disclosure provides amRNA (e.g., mmRNA) encoding a constitutively active IRF7 polypeptidecomprising a deletion of amino acid residues 247-467 (i.e., comprisingamino acid residues 1-246 and 468-503) and further comprising mutationsS475D, S476D, S477D, S479D, S483D and S487D, as set forth in the aminoacid sequence of SEQ ID NO: 18, encoded by the nucleotide sequence shownin SEQ ID NO: 157 or 218.

In other aspects, the disclosure provides a mRNA (e.g., mmRNA) encodinga truncated IRF7 inactive “null” polypeptide construct comprising adeletion of residues 152-246 (i.e., comprising amino acid residues 1-151and 247-503), as set forth in the amino acid sequence of SEQ ID NO: 19,encoded by the nucleotide sequence shown in SEQ ID NO: 158 or 219 (used,for example, for control purposes). In other aspects, the disclosureprovides a mRNA (e.g., mmRNA) encoding a truncated IRF7 inactive “null”polypeptide construct comprising a deletion of residues 1-151 (i.e.,comprising amino acid residues 152-503), as set forth in the amino acidsequence of SEQ ID NO: 20, encoded by the nucleotide sequence shown inSEQ ID NO: 159 or 220 (used, for example, for control purposes).

Additional Immune Potentiator mRNAs that Activate Type I IFN

In addition to the STING and IRF mRNA constructs described above, thedisclosure provides mRNA constructs encoding additional components ofthe Type I IFN signaling pathway that can be use as immune potentiatorsto enhance immune responses through activation of the Type I IFNsignaling pathway. For example, in one embodiment, the immunepotentiator mRNA construct encodes a MyD88 protein. MyD88 is known inthe art to signal upstream of IRF7. In one aspect, the disclosureprovides a mRNA (e.g., mmRNA) encoding a constitutively active MyD88protein, such as mutant MyD88 protein having one or more pointmutations. In one aspect, the disclosure provides a mRNA (e.g., mmRNA)encoding a mutant human or mouse MyD88 protein having a L265Psubstitutions, as set forth in SEQ ID NOs: 75 and 76, respectively.

In another aspect, an immune potentiator mRNA construct encodes a TRAM(TICAM2) protein. TRAM is known in the art to signal upstream of IRF3.In one aspect, the disclosure encompasses a mRNA (e.g., mmRNA) encodinga constitutively active TRAM protein, such as mutant TRAM protein havingone or more point mutations. In another aspect, the disclosureencompasses a wild-type TRAM protein that is overexpressed. In oneaspect, the disclosure provides a mRNA (e.g., mmRNA) encoding a mouseTRAM protein as shown in SEQ ID NO: 77.

In yet other aspects, the disclosure provides an immune potentiator mRNAconstruct encoding a TANK-binding kinase 1 (TBK1) or an inducible IκBkinase (IKKi, also known as IKKε), including constitutively active formsof TBK1 or IKKi, as immune potentiators. TBK1 and IKKi have beendemonstrated to be components of the virus-activated kinase thatphosphorylates IRF3 and IRF7, thus acting upstream from IRF3 and IRF7 inthe Type I IFN signaling pathway (Sharma, S. et al. (2003) Science300:1148-1151). TBK1 and IKKi are involved in the phosphorylation andactivation of transcription factors (e.g. IRF3/7 & NF-κB) that induceexpression of type I IFN genes as well as IFN-inducible genes(Fitzgerald, K. A. et al., (2003) Nat Immunol 4(5):491-496).

Accordingly, in one aspect, the disclosure provides an immunepotentiator mRNA construct that encodes a TBK1 protein, including aconstitutively active form of TBK1, including mutant human TBK1isoforms. In yet other aspects, an immune potentiator mRNA constructencodes a IKKi protein, including a constitutively active form of IKKi,including mutant human IKKi isoforms.

Immune Potentiators mRNAs that Stimulate Inflammatory Responses

In other aspects, the disclosure provides immune potentiator mRNAconstructs that enhance an immune response by stimulating aninflammatory response. Non-limiting examples of agents that stimulate aninflammatory response include STAT1, STAT2, STAT4 and STAT6.Accordingly, the disclosure provides an immune potentiator mRNAconstruct encoding one or a combination of these inflammation-inducingproteins, including a constitutively active form.

Provided herein are mRNAs (e.g., mmRNAs) encoding constitutively activeforms of STAT6, including mutant human STAT6 isoforms for use as immunepotentiators as described herein. mRNAs (e.g., mmRNAs) encodingconstitutively active forms of STAT6, including mutant human STAT6isoforms are set forth in the Sequence Listing herein. The amino acidresidue numbering for mutant human STAT6 polypeptides used hereincorresponds to that used for the 847 amino acid residue wild type humanSTAT6 (isoform 1) available in the art as Genbank Accession NumberNP_001171550.1.

In one embodiment, the disclosure provides a mRNA construct encoding aconstitutively active human STAT6 construct comprising one or more aminoacid mutations selected from the group consisting of S407D, V547A,T548A, Y641F, and combinations thereof. In another embodiment, the mRNAconstruct encodes a constitutively active human STAT6 constructcomprising V547A and T548A mutations, such as the sequence shown in SEQID NO: 78. In another embodiment, the mRNA construct encodes aconstitutively active human STAT6 construct comprising a S407D mutation,such as the sequence shown in SEQ ID NO: 79. In another embodiment, themRNA construct encodes a constitutively active human STAT6 constructcomprising S407D, V547A and T548A mutations, such as the sequence shownin SEQ ID NO: 80. In another embodiment, the mRNA construct encodes aconstitutively active human STAT6 construct comprising V547A, T548A andY641F mutations, such as the sequence shown in SEQ ID NO: 81.

Immune Potentiator mRNAs that Stimulate NFkB Signaling

In other aspects, the disclosure provides immune potentiator mRNAconstructs that enhance an immune response by stimulating an NFkBsignaling, which is known to be involved in stimulation of immuneresponses. Non-limiting examples of proteins that stimulate NFkBsignaling include c-FLIP, IKKβ, RIPK1, Btk and TAK-TAB1. Accordingly, animmune potentiator mRNA construct of the present disclosure can encodeany of these NFkB pathway-inducing proteins, for example in aconstitutively active form.

In one embodiment, the disclosure provides an immune potentiator mRNAconstruct that activates NFκB signaling encodes a c-FLIP (cellularcaspase 8 (FLICE)-like inhibitory protein) protein (also known in theart as CASP8 and FADD-like apoptosis regulator), including aconstitutively active c-FLIP. Provided herein are mRNAs (e.g., mmRNAs)encoding constitutively active forms of c-FLIP, including mutant humanc-FLIP isoforms for use as immune potentiators as described herein.mRNAs (e.g., mmRNAs) encoding constitutively active forms of c-FLIP,including mutant human c-FLIP isoforms are set forth in the SequenceListing herein. The amino acid residue numbering for mutant human c-FLIPpolypeptides used herein corresponds to that used for the 480 amino acidresidue wild type human c-FLIP (isoform 1) available in the art asGenbank Accession Number NP_003870.

In one embodiment, the mRNA encodes a c-FLIP long (L) isoform comprisingtwo DED domains, a p20 domain and a p12 domain, such as having thesequence shown in SEQ ID NO: 82. In another embodiment, the mRNA encodesa c-FLIP short (S) isoform, encoding amino acids 1-227, comprising twoDED domains, such as having the sequence shown in SEQ ID NO: 83. Inanother embodiment, the mRNA encodes a c-FLIP p22 cleavage product,encoding amino acids 1-198, such as having the sequence shown in SEQ IDNO: 84. In another embodiment, the mRNA encodes a c-FLIP p43 cleavageproduct, encoding amino acids 1-376, such as having the sequence shownin SEQ ID NO: 85. In another embodiment, the mRNA encodes a c-FLIP p12cleavage product, encoding amino acids 377-480, such as having thesequence shown in SEQ ID NO: 86.

In another embodiment, an immune potentiator mRNA construct thatactivates NFκB signaling encodes a constitutively active IKKα mRNAconstruct or a constitutively active IKKβ mRNA construct. In oneembodiment, the constitutively active human IKKβ polypeptide comprisesS177E and S181E mutations, such as the sequence shown in SEQ ID NO: 87.In another embodiment, the constitutively active human IKKβ polypeptidecomprises S177A and S181A mutations, such as the sequence shown in SEQID NO: 88. In another embodiment, the mRNA construct encodes aconstitutively active mouse IKKβ polypeptide. In one embodiment, theconstitutively active mouse IKKβ polypeptide comprises S177E and S181Emutations, such as the sequence shown in SEQ ID NO: 148. In anotherembodiment, the constitutively active mouse IKKβ polypeptide comprisesS177A and S181A mutations, such as the sequence shown in SEQ ID NO: 89.In another embodiment, the mRNA construct encodes a constitutivelyactive human or mouse IKKα polypeptide comprising a PEST mutation, suchas having a sequence as shown in SEQ ID NOs: 91-92 (human) or 95-96(mouse). In another embodiment, the mRNA construct encodes aconstitutively active human or mouse IKKβ polypeptide comprising a PESTmutation, such as having the sequence shown in SEQ ID NOs: 93-94 (human)or 97-98 (mouse).

In another embodiment, the disclosure provides an immune potentiatormRNA construct that activates NFκB signaling encoding areceptor-interacting protein kinase 1 (RIPK1) protein. Structure of DNAconstucts encoding RIPK1 constructs that induce immunogenic cell deathare described in the art, for example, Yatim, N. et al. (2015) Science350:328-334 or Orozco, S. et al. (2014) Cell Death Differ. 21:1511-1521,and can be used in the design of suitable RNA constructs that are shownherein to also active NFkB signaling (see Examples). In one embodiment,the mRNA construct encodes RIPK1 amino acids 1-555 of a human or mouseRIPK1 polypeptide as well as an IZ domain, such as having the sequenceshown in SEQ ID N: 99 (human) or 102 (mouse). In one embodiment, themRNA construct encodes RIPK1 amino acids 1-555 of a human or mouse RIPK1polypeptide as well as EE and DM domains, such as having the sequenceshown in SEQ ID NO: 100 (human) or 103 (mouse). In one embodiment, themRNA construct encodes RIPK1 amino acids 1-555 of a human or mouse RIPK1polypeptide as well as RR and DM domains, such as having the sequenceshown in SEQ ID NO: 101 (human) or 104 (mouse).

In yet another embodiment, an immune potentiator mRNA construct thatactivates NFκB signaling encodes a Btk polypeptide, such as a mutant Btkpolypeptide such as a Btk(E41K) polypeptide (e.g., encoding an ORF aminoacid sequence shown in SEQ ID NO: 114)

In yet another embodiment, an immune potentiator mRNA construct thatactivates NFκB signaling encodes a TAK-TAB1 protein, such as aconstitutively active TAK-TAB1.

In one embodiment, an immune potentiator mRNA construct encodes a humanTAK-TAB1 protein, such as having the sequence shown in SEQ ID NO: 105.

Additional Immune Potentiator mRNAs

The present disclosure provides additional immune potentiator mRNAconstructs. For example, in one embodiment, an immune potentiator mRNAconstruct encodes direct IAP binding protein with low pI (DIABLO) (alsoknown as SMAC/DIABLO). As described in the examples herein, DIABLOconstructs induce release of cytokines. In one embodiment, thedisclosure provides a mRNA construct encoding a wild-type human DIABLOIsoform 1 sequence, such as having the sequence shown in SEQ ID NO: 106(corresponding to the 239 amino acid human DIABLO isoform 1 precursordisclosed in the art as Genbank Accession No. NP_063940.1). In anotherembodiment, the mRNA construct encodes a human DIABLO Isoform 1 sequencecomprising an S126L mutation, such as having the sequence shown in SEQID NO: 107. In another embodiment, the mRNA construct encodes aminoacids 56-239 of human DIABLO Isoform 1, such as having the sequenceshown in SEQ ID N: 108. In another embodiment, the mRNA constructencodes amino acids 56-239 of human DIABLO Isoform 1 and comprises anS126L mutation, such as having the sequence shown in SEQ ID NO: 109. Inanother embodiment, the mRNA construct encodes a wild-type human DIABLOIsoform 3 sequence, such as having the sequence shown in SEQ ID NO: 110(corresponding to the 195 amino acid human DIABLO isoform 3 disclosed inthe art as Genbank Accession No. NP_001265271.1). In another embodiment,the mRNA construct encodes a human DIABLO Isoform 3 sequence comprisingan S82L mutation, such as having the sequence shown in SEQ ID NO: 110.In another embodiment, the mRNA construct encodes amino acids 56-195 ofhuman DIABLO Isoform 3, such as having the sequence shown in SEQ ID NO:111. In another embodiment, the mRNA construct encodes amino acids56-195 of human DIABLO Isoform 3 and comprises an S82L mutation, such ashaving the sequence shown in SEQ ID NO: 112.

In additional embodiments, the immune potentiator mRNA construct encodesa SOC3 polypeptide (e.g., encoding an ORF amino acid sequence shown inSEQ ID NO: 115) or encodes a self-activating caspase-1 polypeptide (e.g,encoding any of the ORF amino acid sequences shown in SEQ ID NOs:116-119), which can promote cleavage of pro-IL1β (and pro-IL18 to theirrespective mature forms.

In yet other embodiments, an immune potentiator mRNA construct encodes aprotein that modulates dendritic cell (DC) activity, such as stimulatingDC production, activity or mobilization. A non-limiting example of aprotein that stimulates DC mobilization is FLT3. Accordingly, in oneembodiment, the immune potentiator mRNA construct encodes a FLT3protein.

An immune potentiator mRNA construct typically comprises, in addition tothe polypeptide-encoding sequences, other structural properties asdescribed herein for mRNA constructs (e.g., modified nucleobases, 5′cap, 5′ UTR, 3′ UTR, miR binding site(s), polyA tail, as describedherein). Suitable mRNA construct components are as described herein.

Compositions of Cancer Antigens of Interest and Immune Potentiators

In another aspect, the disclosure provides a composition comprising atleast one messenger RNA (e.g., modified mRNA (mmRNA)) encoding: (i) atleast one antigen of interest (an activating oncogene mutationpeptide(s)); and (ii) a polypeptide that enhances an immune responseagainst the at least one antigen of interest (an activating oncogenemutation peptide(s)) when the at least on mRNA is administered to asubject, wherein said mRNA comprises one or more modified nucleobases.Thus, the disclosure provides compositions comprising an immunepotentiator mRNA and an mRNA encoding an antigen of interest (anactivating oncogene mutation peptide(s)), wherein a single mRNAconstruct can encode both the antigen(s) or interest and the polypeptidethat enhances an immune response to the antigen(s) or, alternatively,the composition can comprise two or more separate mRNA constructs, afirst mRNA and a second mRNA (or third or fourth mRNA), wherein thefirst mRNA encodes the at least one antigen of interest and the secondmRNA encodes the polypeptide that enhances an immune response to theantigen(s) (i.e., the second mRNA comprises the immune potentiator).

In those embodiments comprising a first mRNA encoding an antigen(s) ofinterest and a second mRNA encoding the polypeptide that enhances animmune response to the antigen(s) of interest, the first mRNA and thesecond mRNAs can be coformulated together (e.g., prior tocoadministration), such as coformulated in the same lipid nanoparticle.

In those embodiments comprising a single mRNA encoding both theantigen(s) of interest and the polypeptide that enhances an immuneresponse to the antigen(s) of interest, the sequences encoding thepolypeptide can be positioned on the mRNA construct either upstream ordownstream of the sequences encoding the antigen of interest. Forexample, non-limiting examples of mRNA constructs encoding both anantigen and an immunostimulatory polypeptide include those encoding atleast one mutant KRAS antigen and a constitutively active STINGpolypeptide, e.g., encoding an amino acid sequence shown in any one ofSEQ ID NOs: 48-71. In one embodiment, the constitutively active STINGpolypeptide is located at the N-terminal end of the construct (i.e.,upstream of the antigen-encoding sequences), as shown in SEQ ID NOs:48-57. In another embodiment, the constitutively active STINGpolypeptide is located at the C-terminal end of the construct (i.e.,downstream of the antigen-encoding sequences), as shown in SEQ ID NOs:58-71.

Various mRNAs encoding antigens of interest (e.g., mRNA vaccines) thatcan be used in combination with an immune potentiator mRNA of thedisclosure are described in further detail below.

mRNA Construct Components

An mRNA may be a naturally or non-naturally occurring mRNA. An mRNA mayinclude one or more modified nucleobases, nucleosides, or nucleotides,as described below, in which case it may be referred to as a “modifiedmRNA” or “mmRNA.” As described herein “nucleoside” is defined as acompound containing a sugar molecule (e.g., a pentose or ribose) orderivative thereof in combination with an organic base (e.g., a purineor pyrimidine) or a derivative thereof (also referred to herein as“nucleobase”). As described herein, “nucleotide” is defined as anucleoside including a phosphate group.

An mRNA may include a 5′ untranslated region (5′-UTR), a 3′ untranslatedregion (3′-UTR), and/or a coding region (e.g., an open reading frame).An exemplary 5′ UTR for use in the constructs is shown in SEQ ID NO: 21.An exemplary 3′ UTR for use in the constructs is shown in SEQ ID NO: 22.An exemplary 3′ UTR comprising miR-122 and miR-142.3p binding sites foruse in the constructs is shown in SEQ ID NO: 23. An mRNA may include anysuitable number of base pairs, including tens (e.g., 10, 20, 30, 40, 50,60, 70, 80, 90 or 100), hundreds (e.g., 200, 300, 400, 500, 600, 700,800, or 900) or thousands (e.g., 1000, 2000, 3000, 4000, 5000, 6000,7000, 8000, 9000, 10,000) of base pairs. Any number (e.g., all, some, ornone) of nucleobases, nucleosides, or nucleotides may be an analog of acanonical species, substituted, modified, or otherwise non-naturallyoccurring. In certain embodiments, all of a particular nucleobase typemay be modified.

In some embodiments, an mRNA as described herein may include a 5′ capstructure, a chain terminating nucleotide, optionally a Kozak sequence(also known as a Kozak consensus sequence), a stem loop, a polyAsequence, and/or a polyadenylation signal.

A 5′ cap structure or cap species is a compound including two nucleosidemoieties joined by a linker and may be selected from a naturallyoccurring cap, a non-naturally occurring cap or cap analog, or ananti-reverse cap analog (ARCA). A cap species may include one or moremodified nucleosides and/or linker moieties. For example, a natural mRNAcap may include a guanine nucleotide and a guanine (G) nucleotidemethylated at the 7 position joined by a triphosphate linkage at their5′ positions, e.g., m⁷G(5′)ppp(5′)G, commonly written as m⁷GpppG. A capspecies may also be an anti-reverse cap analog. A non-limiting list ofpossible cap species includes m⁷GpppG, m⁷Gpppm⁷G, m⁷3′dGpppG, m₂^(7,O3′)GpppG, m₂ ^(7,O3′)GppppG, m₂ ^(7,O2′)GppppG, m⁷Gpppm⁷G,m⁷3′dGpppG, m₂ ^(7,O3′)GpppG, m₂ ^(7,O3′)GppppG, and m₂ ^(7,O2′)GppppG.

An mRNA may instead or additionally include a chain terminatingnucleoside. For example, a chain terminating nucleoside may includethose nucleosides deoxygenated at the 2′ and/or 3′ positions of theirsugar group. Such species may include 3′-deoxyadenosine (cordycepin),3′-deoxyuridine, 3′-deoxycytosine, 3′-deoxyguanosine, 3′-deoxythymine,and 2′,3′-dideoxynucleosides, such as 2′,3′-dideoxyadenosine,2′,3′-dideoxyuridine, 2′,3′-dideoxycytosine, 2′,3′-dideoxyguanosine, and2′,3′-dideoxythymine. In some embodiments, incorporation of a chainterminating nucleotide into an mRNA, for example at the 3′-terminus, mayresult in stabilization of the mRNA, as described, for example, inInternational Patent Publication No. WO 2013/103659.

An mRNA may instead or additionally include a stem loop, such as ahistone stem loop. A stem loop may include 2, 3, 4, 5, 6, 7, 8, or morenucleotide base pairs. For example, a stem loop may include 4, 5, 6, 7,or 8 nucleotide base pairs. A stem loop may be located in any region ofan mRNA. For example, a stem loop may be located in, before, or after anuntranslated region (a 5′ untranslated region or a 3′ untranslatedregion), a coding region, or a polyA sequence or tail. In someembodiments, a stem loop may affect one or more function(s) of an mRNA,such as initiation of translation, translation efficiency, and/ortranscriptional termination.

An mRNA may instead or additionally include a polyA sequence and/orpolyadenylation signal. A polyA sequence may be comprised entirely ormostly of adenine nucleotides or analogs or derivatives thereof. A polyAsequence may be a tail located adjacent to a 3′ untranslated region ofan mRNA. In some embodiments, a polyA sequence may affect the nuclearexport, translation, and/or stability of an mRNA.

An mRNA may instead or additionally include a microRNA binding site.

In some embodiments, an mRNA is a bicistronic mRNA comprising a firstcoding region and a second coding region with an intervening sequencecomprising an internal ribosome entry site (IRES) sequence that allowsfor internal translation initiation between the first and second codingregions, or with an intervening sequence encoding a self-cleavingpeptide, such as a 2A peptide. IRES sequences and 2A peptides aretypically used to enhance expression of multiple proteins from the samevector. A variety of IRES sequences are known and available in the artand may be used, including, e.g., the encephalomyocarditis virus IRES.

In one embodiment, the polynucleotides of the present disclosure mayinclude a sequence encoding a self-cleaving peptide. The self-cleavingpeptide may be, but is not limited to, a 2A peptide. A variety of 2Apeptides are known and available in the art and may be used, includinge.g., the foot and mouth disease virus (FMDV) 2A peptide, the equinerhinitis A virus 2A peptide, the Thosea asigna virus 2A peptide, and theporcine teschovirus-1 2A peptide. 2A peptides are used by severalviruses to generate two proteins from one transcript byribosome-skipping, such that a normal peptide bond is impaired at the 2Apeptide sequence, resulting in two discontinuous proteins being producedfrom one translation event. As a non-limiting example, the 2A peptidemay have the protein sequence: GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 24),fragments or variants thereof. In one embodiment, the 2A peptide cleavesbetween the last glycine and last proline. As another non-limitingexample, the polynucleotides of the present disclosure may include apolynucleotide sequence encoding the 2A peptide having the proteinsequence GSGATNFSLLKQAGDVEENPGP (SEQ ID NO:24) fragments or variantsthereof. One example of a polynucleotide sequence encoding the 2Apeptide is: GGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCT (SEQ ID NO: 25). In one illustrative embodiment, a 2Apeptide is encoded by the following sequence:5′-TCCGGACTCAGATCCGGGGATCTCAAAATTGTCGCTCCTGTCAAACAAACTCTTAACTTTGATTTACTCAAACTGGCTGGGGATGTAGAAAGCAATCCAGGTCCACTC-3′(SEQ ID NO: 26).The polynucleotide sequence of the 2A peptide may be modified or codonoptimized by the methods described herein and/or are known in the art.

In one embodiment, this sequence may be used to separate the codingregions of two or more polypeptides of interest. As a non-limitingexample, the sequence encoding the F2A peptide may be between a firstcoding region A and a second coding region B (A-F2Apep-B). The presenceof the F2A peptide results in the cleavage of the one long proteinbetween the glycine and the proline at the end of the F2A peptidesequence (NPGP is cleaved to result in NPG and P) thus creating separateprotein A (with 21 amino acids of the F2A peptide attached, ending withNPG) and separate protein B (with 1 amino acid, P, of the F2A peptideattached). Likewise, for other 2A peptides (P2A, T2A and E2A), thepresence of the peptide in a long protein results in cleavage betweenthe glycine and proline at the end of the 2A peptide sequence (NPGP iscleaved to result in NPG and P). Protein A and protein B may be the sameor different peptides or polypeptides of interest. In particularembodiments, protein A is a polypeptide that induces immunogenic celldeath and protein B is another polypeptide that stimulates aninflammatory and/or immune response and/or regulates immuneresponsiveness (as described further below).

Modified mRNAs

In some embodiments, an mRNA of the disclosure comprises one or moremodified nucleobases, nucleosides, or nucleotides (termed “modifiedmRNAs” or “mmRNAs”). In some embodiments, modified mRNAs may have usefulproperties, including enhanced stability, intracellular retention,enhanced translation, and/or the lack of a substantial induction of theinnate immune response of a cell into which the mRNA is introduced, ascompared to a reference unmodified mRNA. Therefore, use of modifiedmRNAs may enhance the efficiency of protein production, intracellularretention of nucleic acids, as well as possess reduced immunogenicity.

In some embodiments, an mRNA includes one or more (e.g., 1, 2, 3 or 4)different modified nucleobases, nucleosides, or nucleotides. In someembodiments, an mRNA includes one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more) different modifiednucleobases, nucleosides, or nucleotides. In some embodiments, themodified mRNA may have reduced degradation in a cell into which the mRNAis introduced, relative to a corresponding unmodified mRNA.

In some embodiments, the modified nucleobase is a modified uracil.Exemplary nucleobases and nucleosides having a modified uracil includepseudouridine (ψ), pyridin-4-one ribonucleoside, 5-aza-uridine,6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s²U),4-thio-uridine (s⁴U), 4-thio-pseudouridine, 2-thio-pseudouridine,5-hydroxy-uridine (ho⁵U), 5-aminoallyl-uridine, 5-halo-uridine (e.g.,5-iodo-uridineor 5-bromo-uridine), 3-methyl-uridine (m³U),5-methoxy-uridine (mo⁵U), uridine 5-oxyacetic acid (cmo⁵U), uridine5-oxyacetic acid methyl ester (mcmo⁵U), 5-carboxymethyl-uridine (cm⁵U),1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm⁵U),5-carboxyhydroxymethyl-uridine methyl ester (mchm⁵U),5-methoxycarbonylmethyl-uridine (mcm⁵U),5-methoxycarbonylmethyl-2-thio-uridine (mcm⁵s²U),5-aminomethyl-2-thio-uridine (nm⁵s²U), 5-methylaminomethyl-uridine(mnm⁵U), 5-methylaminomethyl-2-thio-uridine (mnm⁵s²U),5-methylaminomethyl-2-seleno-uridine (mnm⁵se²U),5-carbamoylmethyl-uridine (ncm⁵U), 5-carboxymethylaminomethyl-uridine(cmnm⁵U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm⁵s²U),5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine(τm⁵U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine(τm⁵s²U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m⁵U,i.e., having the nucleobase deoxythymine), 1-methyl-pseudouridine (m¹ψ),5-methyl-2-thio-uridine (m⁵s²U), 1-methyl-4-thio-pseudouridine (m¹s⁴ψ),4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m³ψ),2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D),dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m⁵D),2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine,2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine,4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine,3-(3-amino-3-carboxypropyl)uridine (acp³U),1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp³ ψ),5-(isopentenylaminomethyl)uridine (inm⁵U),5-(isopentenylaminomethyl)-2-thio-uridine (inm⁵s²U), α-thio-uridine,2′-O-methyl-uridine (Um), 5,2′-O-dimethyl-uridine (m⁵Um),2′-O-methyl-pseudouridine (ψm), 2-thio-2′-O-methyl-uridine (s²Um),5-methoxycarbonylmethyl-2′-O-methyl-uridine (mcm⁵Um),5-carbamoylmethyl-2′-O-methyl-uridine (ncm⁵Um),5-carboxymethylaminomethyl-2′-O-methyl-uridine (cmnm⁵Um),3,2′-O-dimethyl-uridine (m³Um), and5-(isopentenylaminomethyl)-2′-O-methyl-uridine (inm⁵Um), 1-thio-uridine,deoxythymidine, 2′-F-ara-uridine, 2′-F-uridine, 2′-OH-ara-uridine,5-(2-carbomethoxyvinyl) uridine, and 5-[3-(1-E-propenylamino)]uridine.

In some embodiments, the modified nucleobase is a modified cytosine.Exemplary nucleobases and nucleosides having a modified cytosine include5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine(m³C), N4-acetyl-cytidine (ac⁴C), 5-formyl-cytidine (f⁵C),N4-methyl-cytidine (m⁴C), 5-methyl-cytidine (m⁵C), 5-halo-cytidine(e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm⁵C),1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine,2-thio-cytidine (s²C), 2-thio-5-methyl-cytidine,4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine,4-thio-1-methyl-1-deaza-pseudoisocytidine,1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine,lysidine (k₂C), α-thio-cytidine, 2′-O-methyl-cytidine (Cm),5,2′-O-dimethyl-cytidine (m⁵Cm), N4-acetyl-2′-O-methyl-cytidine (ac⁴Cm),N4,2′-O-dimethyl-cytidine (m⁴Cm), 5-formyl-2′-O-methyl-cytidine (f⁵Cm),N4,N4,2′-O-trimethyl-cytidine (m⁴ ₂Cm), 1-thio-cytidine,2′-F-ara-cytidine, 2′-F-cytidine, and 2′-OH-ara-cytidine.

In some embodiments, the modified nucleobase is a modified adenine.Exemplary nucleobases and nucleosides having a modified adenine includeα-thio-adenosine, 2-amino-purine, 2, 6-diaminopurine,2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine(e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine,7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine,7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine,7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine (m¹A),2-methyl-adenine (m²A), N6-methyl-adenosine (m⁶A),2-methylthio-N6-methyl-adenosine (ms²m⁶A), N6-isopentenyl-adenosine(i⁶A), 2-methylthio-N6-isopentenyl-adenosine (ms²i⁶A),N6-(cis-hydroxyisopentenyl)adenosine (io⁶A),2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms²io⁶A),N6-glycinylcarbamoyl-adenosine (g⁶A), N6-threonylcarbamoyl-adenosine(t⁶A), N6-methyl-N6-threonylcarbamoyl-adenosine (m⁶t⁶A),2-methylthio-N6-threonylcarbamoyl-adenosine (ms²g⁶A),N6,N6-dimethyl-adenosine (m⁶ ₂A), N6-hydroxynorvalylcarbamoyl-adenosine(hn⁶A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms²hn⁶A),N6-acetyl-adenosine (ac⁶A), 7-methyl-adenine, 2-methylthio-adenine,2-methoxy-adenine, α-thio-adenosine, 2′-O-methyl-adenosine (Am),N6,2′-O-dimethyl-adenosine (m⁶Am), N6,N6,2′-O-trimethyl-adenosine (m⁶₂Am), 1,2′-O-dimethyl-adenosine (m¹Am), 2′-O-ribosyladenosine(phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine,8-azido-adenosine, 2′-F-ara-adenosine, 2′-F-adenosine,2′-OH-ara-adenosine, and N6-(19-amino-pentaoxanonadecyl)-adenosine.

In some embodiments, the modified nucleobase is a modified guanine.Exemplary nucleobases and nucleosides having a modified guanine includeα-thio-guanosine, inosine (I), 1-methyl-inosine (m¹I), wyosine (imG),methylwyosine (mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2),wybutosine (yW), peroxywybutosine (o₂yW), hydroxywybutosine (OhyW),undermodified hydroxywybutosine (OhyW*), 7-deaza-guanosine, queuosine(Q), epoxyqueuosine (oQ), galactosyl-queuosine (galQ),mannosyl-queuosine (manQ), 7-cyano-7-deaza-guanosine (preQ₀),7-aminomethyl-7-deaza-guanosine (preQ₁), archaeosine (G⁺),7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine,6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m⁷G),6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine,1-methyl-guanosine (m¹G), N2-methyl-guanosine (m²G),N2,N2-dimethyl-guanosine (m² ₂G), N2,7-dimethyl-guanosine (m^(2,7)G),N2, N2,7-dimethyl-guanosine (m^(2,2,7)G), 8-oxo-guanosine,7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine,N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine,α-thio-guanosine, 2′-O-methyl-guanosine (Gm),N2-methyl-2′-O-methyl-guanosine (m²Gm),N2,N2-dimethyl-2′-O-methyl-guanosine (m² ₂Gm),1-methyl-2′-O-methyl-guanosine (mGm),N2,7-dimethyl-2′-O-methyl-guanosine (m^(2,7)Gm), 2′-O-methyl-inosine(Im), 1,2′-O-dimethyl-inosine (m¹Im), 2′-O-ribosylguanosine (phosphate)(Gr(p)), 1-thio-guanosine, 06-methyl-guanosine, 2′-F-ara-guanosine, and2′-F-guanosine.

In some embodiments, an mRNA of the disclosure includes a combination ofone or more of the aforementioned modified nucleobases (e.g., acombination of 2, 3 or 4 of the aforementioned modified nucleobases.)

In some embodiments, the modified nucleobase is pseudouridine (ψ),N1-methylpseudouridine (m¹ψ), 2-thiouridine, 4′-thiouridine,5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine,2-thio-dihydropseudouridine, 2-thio-dihydrouridine,2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine,4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine,4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine,5-methoxyuridine, or 2′-O-methyl uridine. In some embodiments, an mRNAof the disclosure includes a combination of one or more of theaforementioned modified nucleobases (e.g., a combination of 2, 3 or 4 ofthe aforementioned modified nucleobases.) In one embodiment, themodified nucleobase is N1-methylpseudouridine (m¹ψ) and the mRNA of thedisclosure is fully modified with N1-methylpseudouridine (m¹ψ). In someembodiments, N1-methylpseudouridine (m¹ψ) represents from 75-100% of theuracils in the mRNA. In some embodiments, N1-methylpseudouridine (m¹ψ)represents 100% of the uracils in the mRNA.

In some embodiments, the modified nucleobase is a modified cytosine.Exemplary nucleobases and nucleosides having a modified cytosine includeN4-acetyl-cytidine (ac⁴C), 5-methyl-cytidine (m⁵C), 5-halo-cytidine(e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm⁵C),1-methyl-pseudoisocytidine, 2-thio-cytidine (s²C),2-thio-5-methyl-cytidine. In some embodiments, an mRNA of the disclosureincludes a combination of one or more of the aforementioned modifiednucleobases (e.g., a combination of 2, 3 or 4 of the aforementionedmodified nucleobases.)

In some embodiments, the modified nucleobase is a modified adenine.Exemplary nucleobases and nucleosides having a modified adenine include7-deaza-adenine, 1-methyl-adenosine (m¹A), 2-methyl-adenine (m²A),N6-methyl-adenosine (m⁶A). In some embodiments, an mRNA of thedisclosure includes a combination of one or more of the aforementionedmodified nucleobases (e.g., a combination of 2, 3 or 4 of theaforementioned modified nucleobases.)

In some embodiments, the modified nucleobase is a modified guanine.Exemplary nucleobases and nucleosides having a modified guanine includeinosine (I), 1-methyl-inosine (m¹I), wyosine (imG), methylwyosine(mimG), 7-deaza-guanosine, 7-cyano-7-deaza-guanosine (preQ₀),7-aminomethyl-7-deaza-guano sine (preQ₁), 7-methyl-guanosine (m⁷G),1-methyl-guanosine (m¹G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine. Insome embodiments, an mRNA of the disclosure includes a combination ofone or more of the aforementioned modified nucleobases (e.g., acombination of 2, 3 or 4 of the aforementioned modified nucleobases.)

In some embodiments, the modified nucleobase is 1-methyl-pseudouridine(m¹ψ), 5-methoxy-uridine (mo⁵U), 5-methyl-cytidine (m⁵C), pseudouridine(ψ), α-thio-guanosine, or α-thio-adenosine. In some embodiments, an mRNAof the disclosure includes a combination of one or more of theaforementioned modified nucleobases (e.g., a combination of 2, 3 or 4 ofthe aforementioned modified nucleobases.)

In some embodiments, the mRNA comprises pseudouridine (ψ). In someembodiments, the mRNA comprises pseudouridine (ψ) and 5-methyl-cytidine(m⁵C). In some embodiments, the mRNA comprises 1-methyl-pseudouridine(m¹ψ). In some embodiments, the mRNA comprises 1-methyl-pseudouridine(m¹ψ) and 5-methyl-cytidine (m⁵C). In some embodiments, the mRNAcomprises 2-thiouridine (s²U). In some embodiments, the mRNA comprises2-thiouridine and 5-methyl-cytidine (m⁵C). In some embodiments, the mRNAcomprises 5-methoxy-uridine (mo⁵U). In some embodiments, the mRNAcomprises 5-methoxy-uridine (mo⁵U) and 5-methyl-cytidine (m⁵C). In someembodiments, the mRNA comprises 2′-O-methyl uridine. In someembodiments, the mRNA comprises 2′-O-methyl uridine and5-methyl-cytidine (m⁵C). In some embodiments, the mRNA comprisescomprises N6-methyl-adenosine (m⁶A). In some embodiments, the mRNAcomprises N6-methyl-adenosine (m⁶A) and 5-methyl-cytidine (m⁵C).

In certain embodiments, an mRNA of the disclosure is uniformly modified(i.e., fully modified, modified through-out the entire sequence) for aparticular modification. For example, an mRNA can be uniformly modifiedwith N1-methylpseudouridine (m¹ψ) or 5-methyl-cytidine (m⁵C), meaningthat all uridines or all cytosine nucleosides in the mRNA sequence arereplaced with N1-methylpseudouridine (m¹ψ) or 5-methyl-cytidine (m⁵C).Similarly, mRNAs of the disclosure can be uniformly modified for anytype of nucleoside residue present in the sequence by replacement with amodified residue such as those set forth above.

In some embodiments, an mRNA of the disclosure may be modified in acoding region (e.g., an open reading frame encoding a polypeptide). Inother embodiments, an mRNA may be modified in regions besides a codingregion. For example, in some embodiments, a 5′-UTR and/or a 3′-UTR areprovided, wherein either or both may independently contain one or moredifferent nucleoside modifications. In such embodiments, nucleosidemodifications may also be present in the coding region.

Examples of nucleoside modifications and combinations thereof that maybe present in mmRNAs of the present disclosure include, but are notlimited to, those described in PCT Patent Application Publications:WO2012045075, WO2014081507, WO2014093924, WO2014164253, andWO2014159813.

The mmRNAs of the disclosure can include a combination of modificationsto the sugar, the nucleobase, and/or the internucleoside linkage. Thesecombinations can include any one or more modifications described herein.

Examples of modified nucleosides and modified nucleoside combinationsare provided below in Table 1 and Table 2. These combinations ofmodified nucleotides can be used to form the mmRNAs of the disclosure.In certain embodiments, the modified nucleosides may be partially orcompletely substituted for the natural nucleotides of the mRNAs of thedisclosure. As a non-limiting example, the natural nucleotide uridinemay be substituted with a modified nucleoside described herein. Inanother non-limiting example, the natural nucleoside uridine may bepartially substituted (e.g., about 0.1%, 1%, 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or99.9% of the natural uridines) with at least one of the modifiednucleoside disclosed herein.

TABLE 1 Combinations of Nucleoside Modifications Modified NucleotideModified Nucleotide Combination α-thio-cytidineα-thio-cytidine/5-iodo-uridine α-thio-cytidine/N1-methyl-pseudouridineα-thio-cytidine/α-thio-uridine α-thio-cytidine/5-methyl-uridineα-thio-cytidine/pseudo-uridine about 50% of the cytosines areα-thio-cytidine pseudoisocytidine pseudoisocytidine/5-iodo-uridinepseudoisocytidine/N1-methyl-pseudouridinepseudoisocytidine/α-thio-uridine pseudoisocytidine/5-methyl-uridinepseudoisocytidine/pseudouridine about 25% of cytosines arepseudoisocytidine pseudoisocytidine/about 50% of uridines are N1-methyl-pseudouridine and about 50% of uridines are pseudouridinepseudoisocytidine/about 25% of uridines are N1- methyl-pseudouridine andabout 25% of uridines are pseudouridine pyrrolo-cytidinepyrrolo-cytidine/5-iodo-uridine pyrrolo-cytidine/N1-methyl-pseudouridinepyrrolo-cytidine/α-thio-uridine pyrrolo-cytidine/5-methyl-uridinepyrrolo-cytidine/pseudouridine about 50% of the cytosines arepyrrolo-cytidine 5-methyl-cytidine 5-methyl-cytidine/5-iodo-uridine5-methyl-cytidine/N1-methyl-pseudouridine5-methyl-cytidine/α-thio-uridine 5-methyl-cytidine/5-methyl-uridine5-methyl-cytidine/pseudouridine about 25% of cytosines are5-methyl-cytidine about 50% of cytosines are 5-methyl-cytidine5-methyl-cytidine/5-methoxy-uridine 5-methyl-cytidine/5-bromo-uridine5-methyl-cytidine/2-thio-uridine 5-methyl-cytidine/about 50% of uridinesare 2- thio-uridine about 50% of uridines are 5-methyl-cytidine/about50% of uridines are 2-thio-uridine N4-acetyl-cytidineN4-acetyl-cytidine/5-iodo-uridineN4-acetyl-cytidine/N1-methyl-pseudouridineN4-acetyl-cytidine/α-thio-uridine N4-acetyl-cytidine/5-methyl-uridineN4-acetyl-cytidine/pseudouridine about 50% of cytosines areN4-acetyl-cytidine about 25% of cytosines are N4-acetyl-cytidineN4-acetyl-cytidine/5-methoxy-uridine N4-acetyl-cytidine/5-bromo-uridineN4-acetyl-cytidine/2-thio-uridine about 50% of cytosines areN4-acetyl-cytidine/ about 50% of uridines are 2-thio-uridine

TABLE 2 Modified Nucleosides and Combinations Thereof1-(2,2,2-Trifluoroethyl)pseudo-UTP 1-Ethyl-pseudo-UTP1-Methyl-pseudo-U-alpha-thio-TP 1-methyl-pseudouridine TP, ATP, GTP, CTP1-methyl-pseudo-UTP/5-methyl-CTP/ATP/GTP 1-methyl-pseudo-UTP/CTP/ATP/GTP1-Propyl-pseudo-UTP 25% 5-Aminoallyl-CTP + 75% CTP/25% 5-Methoxy-UTP +75% UTP 25% 5-Aminoallyl-CTP + 75% CTP/75% 5-Methoxy-UTP + 25% UTP 25%5-Bromo-CTP + 75% CTP/25% 5-Methoxy-UTP + 75% UTP 25% 5-Bromo-CTP + 75%CTP/75% 5-Methoxy-UTP + 25% UTP 25% 5-Bromo-CTP + 75%CTP/1-Methyl-pseudo-UTP 25% 5-Carboxy-CTP + 75% CTP/25% 5-Methoxy-UTP +75% UTP 25% 5-Carboxy-CTP + 75% CTP/75% 5-Methoxy-UTP + 25% UTP 25%5-Ethyl-CTP + 75% CTP/25% 5-Methoxy-UTP + 75% UTP 25% 5-Ethyl-CTP + 75%CTP/75% 5-Methoxy-UTP + 25% UTP 25% 5-Ethynyl-CTP + 75% CTP/25%5-Methoxy-UTP + 75% UTP 25% 5-Ethynyl-CTP + 75% CTP/75% 5-Methoxy-UTP +25% UTP 25% 5-Fluoro-CTP + 75% CTP/25% 5-Methoxy-UTP + 75% UTP 25%5-Fluoro-CTP + 75% CTP/75% 5-Methoxy-UTP + 25% UTP 25% 5-Formyl-CTP +75% CTP/25% 5-Methoxy-UTP + 75% UTP 25% 5-Formyl-CTP + 75% CTP/75%5-Methoxy-UTP + 25% UTP 25% 5-Hydroxymethyl-CTP + 75% CTP/25%5-Methoxy-UTP + 75% UTP 25% 5-Hydroxymethyl-CTP + 75% CTP/75%5-Methoxy-UTP + 25% UTP 25% 5-Iodo-CTP + 75% CTP/25% 5-Methoxy-UTP + 75%UTP 25% 5-Iodo-CTP + 75% CTP/75% 5-Methoxy-UTP + 25% UTP 25%5-Methoxy-CTP + 75% CTP/25% 5-Methoxy-UTP + 75% UTP 25% 5-Methoxy-CTP +75% CTP/75% 5-Methoxy-UTP + 25% UTP 25% 5-Methyl-CTP + 75% CTP/25%5-Methoxy-UTP + 75% 1-Methyl- pseudo-UTP 25% 5-Methyl-CTP + 75% CTP/25%5-Methoxy-UTP + 75% UTP 25% 5-Methyl-CTP + 75% CTP/50% 5-Methoxy-UTP +50% 1-Methyl- pseudo-UTP 25% 5-Methyl-CTP + 75% CTP/50% 5-Methoxy-UTP +50% UTP 25% 5-Methyl-CTP + 75% CTP/5-Methoxy-UTP 25% 5-Methyl-CTP + 75%CTP/75% 5-Methoxy-UTP + 25% 1-Methyl- pseudo-UTP 25% 5-Methyl-CTP + 75%CTP/75% 5-Methoxy-UTP + 25% UTP 25% 5-Phenyl-CTP + 75% CTP/25%5-Methoxy-UTP + 75% UTP 25% 5-Phenyl-CTP + 75% CTP/75% 5-Methoxy-UTP +25% UTP 25% 5-Trifluoromethyl-CTP + 75% CTP/25% 5-Methoxy-UTP + 75% UTP25% 5-Trifluoromethyl-CTP + 75% CTP/75% 5-Methoxy-UTP + 25% UTP 25%5-Trifluoromethyl-CTP + 75% CTP/1-Methyl-pseudo-UTP 25% N4-Ac-CTP + 75%CTP/25% 5-Methoxy-UTP + 75% UTP 25% N4-Ac-CTP + 75% CTP/75%5-Methoxy-UTP + 25% UTP 25% N4-Bz-CTP + 75% CTP/25% 5-Methoxy-UTP + 75%UTP 25% N4-Bz-CTP + 75% CTP/75% 5-Methoxy-UTP + 25% UTP 25%N4-Methyl-CTP + 75% CTP/25% 5-Methoxy-UTP + 75% UTP 25% N4-Methyl-CTP +75% CTP/75% 5-Methoxy-UTP + 25% UTP 25% Pseudo-iso-CTP + 75% CTP/25%5-Methoxy-UTP + 75% UTP 25% Pseudo-iso-CTP + 75% CTP/75% 5-Methoxy-UTP +25% UTP 25% 5-Bromo-CTP/75% CTP/Pseudo-UTP 25% 5-methoxy-UTP/25%5-methyl-CTP/ATP/GTP 25% 5-methoxy-UTP/5-methyl-CTP/ATP/GTP 25%5-methoxy-UTP/75% 5-methyl-CTP/ATP/GTP 25% 5-methoxy-UTP/CTP/ATP/GTP 25%5-metoxy-UTP/50% 5-methyl-CTP/ATP/GTP 2-Amino-ATP 2-Thio-CTP2-thio-pseudouridine TP, ATP, GTP, CTP 2-Thio-pseudo-UTP 2-Thio-UTP3-Methyl-CTP 3-Methyl-pseudo-UTP 4-Thio-UTP 50% 5-Bromo-CTP + 50%CTP/1-Methyl-pseudo-UTP 50% 5-Hydroxymethyl-CTP + 50%CTP/1-Methyl-pseudo-UTP 50% 5-methoxy-UTP/5-methyl-CTP/ATP/GTP 50%5-Methyl-CTP + 50% CTP/25% 5-Methoxy-UTP + 75% 1-Methyl- pseudo-UTP 50%5-Methyl-CTP + 50% CTP/25% 5-Methoxy-UTP + 75% UTP 50% 5-Methyl-CTP +50% CTP/50% 5-Methoxy-UTP + 50% 1-Methyl- pseudo-UTP 50% 5-Methyl-CTP +50% CTP/50% 5-Methoxy-UTP + 50% UTP 50% 5-Methyl-CTP + 50%CTP/5-Methoxy-UTP 50% 5-Methyl-CTP + 50% CTP/75% 5-Methoxy-UTP + 25%1-Methyl- pseudo-UTP 50% 5-Methyl-CTP + 50% CTP/75% 5-Methoxy-UTP + 25%UTP 50% 5-Trifluoromethyl-CTP + 50% CTP/1-Methyl-pseudo-UTP 50%5-Bromo-CTP/50% CTP/Pseudo-UTP 50% 5-methoxy-UTP/25%5-methyl-CTP/ATP/GTP 50% 5-methoxy-UTP/50% 5-methyl-CTP/ATP/GTP 50%5-methoxy-UTP/75% 5-methyl-CTP/ATP/GTP 50% 5-methoxy-UTP/CTP/ATP/GTP5-Aminoallyl-CTP 5-Aminoallyl-CTP/5-Methoxy-UTP 5-Aminoallyl-UTP5-Bromo-CTP 5-Bromo-CTP/5-Methoxy-UTP 5-Bromo-CTP/1-Methyl-pseudo-UTP5-Bromo-CTP/Pseudo-UTP 5-bromocytidine TP, ATP, GTP, UTP 5-Bromo-UTP5-Carboxy-CTP/5-Methoxy-UTP 5-Ethyl-CTP/5-Methoxy-UTP5-Ethynyl-CTP/5-Methoxy-UTP 5-Fluoro-CTP/5-Methoxy-UTP5-Formyl-CTP/5-Methoxy-UTP 5-Hydroxy-methyl-CTP/5-Methoxy-UTP5-Hydroxymethyl-CTP 5-Hydroxymethyl-CTP/1-Methyl-pseudo-UTP5-Hydroxymethyl-CTP/5-Methoxy-UTP 5-hydroxymethyl-cytidine TP, ATP, GTP,UTP 5-Iodo-CTP/5-Methoxy-UTP 5-Me-CTP/5-Methoxy-UTP 5-Methoxy carbonylmethyl-UTP 5-Methoxy-CTP/5-Methoxy-UTP 5-methoxy-uridine TP, ATP, GTP,UTP 5-methoxy-UTP 5-Methoxy-UTP 5-Methoxy-UTP/N6-Isopentenyl-ATP5-methoxy-UTP/25% 5-methyl-CTP/ATP/GTP5-methoxy-UTP/5-methyl-CTP/ATP/GTP 5-methoxy-UTP/75%5-methyl-CTP/ATP/GTP 5-methoxy-UTP/CTP/ATP/GTP 5-Methyl-2-thio-UTP5-Methylaminomethyl-UTP 5-Methyl-CTP/5-Methoxy-UTP5-Methyl-CTP/5-Methoxy-UTP(cap 0) 5-Methyl-CTP/5-Methoxy-UTP(No cap)5-Methyl-CTP/25% 5-Methoxy-UTP + 75% 1-Methyl-pseudo-UTP5-Methyl-CTP/25% 5-Methoxy-UTP + 75% UTP 5-Methyl-CTP/50%5-Methoxy-UTP + 50% 1-Methyl-pseudo-UTP 5-Methyl-CTP/50% 5-Methoxy-UTP +50% UTP 5-Methyl-CTP/5-Methoxy-UTP/N6-Me-ATP 5-Methyl-CTP/75%5-Methoxy-UTP + 25% 1-Methyl-pseudo-UTP 5-Methyl-CTP/75% 5-Methoxy-UTP +25% UTP 5-Phenyl-CTP/5-Methoxy-UTP 5-Trifluoro-methyl-CTP/5-Methoxy-UTP5-Trifluoromethyl-CTP 5-Trifluoromethyl-CTP/5-Methoxy-UTP5-Trifluoromethyl-CTP/1-Methyl-pseudo-UTP5-Trifluoromethyl-CTP/Pseudo-UTP 5-Trifluoromethyl-UTP5-trifluromethylcytidine TP, ATP, GTP, UTP 75% 5-Aminoallyl-CTP + 25%CTP/25% 5-Methoxy-UTP + 75% UTP 75% 5-Aminoallyl-CTP + 25% CTP/75%5-Methoxy-UTP + 25% UTP 75% 5-Bromo-CTP + 25% CTP/25% 5-Methoxy-UTP +75% UTP 75% 5-Bromo-CTP + 25% CTP/75% 5-Methoxy-UTP + 25% UTP 75%5-Carboxy-CTP + 25% CTP/25% 5-Methoxy-UTP + 75% UTP 75% 5-Carboxy-CTP +25% CTP/75% 5-Methoxy-UTP + 25% UTP 75% 5-Ethyl-CTP + 25% CTP/25%5-Methoxy-UTP + 75% UTP 75% 5-Ethyl-CTP + 25% CTP/75% 5-Methoxy-UTP +25% UTP 75% 5-Ethynyl-CTP + 25% CTP/25% 5-Methoxy-UTP + 75% UTP 75%5-Ethynyl-CTP + 25% CTP/75% 5-Methoxy-UTP + 25% UTP 75% 5-Fluoro-CTP +25% CTP/25% 5-Methoxy-UTP + 75% UTP 75% 5-Fluoro-CTP + 25% CTP/75%5-Methoxy-UTP + 25% UTP 75% 5-Formyl-CTP + 25% CTP/25% 5-Methoxy-UTP +75% UTP 75% 5-Formyl-CTP + 25% CTP/75% 5-Methoxy-UTP + 25% UTP 75%5-Hydroxymethyl-CTP + 25% CTP/25% 5-Methoxy-UTP + 75% UTP 75%5-Hydroxymethyl-CTP + 25% CTP/75% 5-Methoxy-UTP + 25% UTP 75%5-Iodo-CTP + 25% CTP/25% 5-Methoxy-UTP + 75% UTP 75% 5-Iodo-CTP + 25%CTP/75% 5-Methoxy-UTP + 25% UTP 75% 5-Methoxy-CTP + 25% CTP/25%5-Methoxy-UTP + 75% UTP 75% 5-Methoxy-CTP + 25% CTP/75% 5-Methoxy-UTP +25% UTP 75% 5-methoxy-UTP/5-methyl-CTP/ATP/GTP 75% 5-Methyl-CTP + 25%CTP/25% 5-Methoxy-UTP + 75% 1-Methyl- pseudo-UTP 75% 5-Methyl-CTP + 25%CTP/25% 5-Methoxy-UTP + 75% UTP 75% 5-Methyl-CTP + 25% CTP/50%5-Methoxy-UTP + 50% 1-Methyl- pseudo-UTP 75% 5-Methyl-CTP + 25% CTP/50%5-Methoxy-UTP + 50% UTP 75% 5-Methyl-CTP + 25% CTP/5-Methoxy-UTP 75%5-Methyl-CTP + 25% CTP/75% 5-Methoxy-UTP + 25% 1-Methyl- pseudo-UTP 75%5-Methyl-CTP + 25% CTP/75% 5-Methoxy-UTP + 25% UTP 75% 5-Phenyl-CTP +25% CTP/25% 5-Methoxy-UTP + 75% UTP 75% 5-Phenyl-CTP + 25% CTP/75%5-Methoxy-UTP + 25% UTP 75% 5-Trifluoromethyl-CTP + 25% CTP/25%5-Methoxy-UTP + 75% UTP 75% 5-Trifluoromethyl-CTP + 25% CTP/75%5-Methoxy-UTP + 25% UTP 75% 5-Trifluoromethyl-CTP + 25%CTP/1-Methyl-pseudo-UTP 75% N4-Ac-CTP + 25% CTP/25% 5-Methoxy-UTP + 75%UTP 75% N4-Ac-CTP + 25% CTP/75% 5-Methoxy-UTP + 25% UTP 75% N4-Bz-CTP +25% CTP/25% 5-Methoxy-UTP + 75% UTP 75% N4-Bz-CTP + 25% CTP/75%5-Methoxy-UTP + 25% UTP 75% N4-Methyl-CTP + 25% CTP/25% 5-Methoxy-UTP +75% UTP 75% N4-Methyl-CTP + 25% CTP/75% 5-Methoxy-UTP + 25% UTP 75%Pseudo-iso-CTP + 25% CTP/25% 5-Methoxy-UTP + 75% UTP 75%Pseudo-iso-CTP + 25% CTP/75% 5-Methoxy-UTP + 25% UTP 75% 5-Bromo-CTP/25%CTP/1-Methyl-pseudo-UTP 75% 5-Bromo-CTP/25% CTP/Pseudo-UTP 75%5-methoxy-UTP/25% 5-methyl-CTP/ATP/GTP 75% 5-methoxy-UTP/50%5-methyl-CTP/ATP/GTP 75% 5-methoxy-UTP/75% 5-methyl-CTP/ATP/GTP 75%5-methoxy-UTP/CTP/ATP/GTP 8-Aza-ATP Alpha-thio-CTP CTP/25%5-Methoxy-UTP + 75% 1-Methyl-pseudo-UTP CTP/25% 5-Methoxy-UTP + 75% UTPCTP/50% 5-Methoxy-UTP + 50% 1-Methyl-pseudo-UTP CTP/50% 5-Methoxy-UTP +50% UTP CTP/5-Methoxy-UTP CTP/5-Methoxy-UTP (cap 0) CTP/5-Methoxy-UTP(Nocap) CTP/75% 5-Methoxy-UTP + 25% 1-Methyl-pseudo-UTP CTP/75%5-Methoxy-UTP + 25% UTP CTP/UTP(No cap) N1-Me-GTP N4-Ac-CTPN4Ac-CTP/1-Methyl-pseudo-UTP N4Ac-CTP/5-Methoxy-UTP N4-acetyl-cytidineTP, ATP, GTP, UTP N4-Bz-CTP/5-Methoxy-UTP N4-methyl CTPN4-Methyl-CTP/5-Methoxy-UTP Pseudo-iso-CTP/5-Methoxy-UTPPseudoU-alpha-thio-TP pseudouridine TP, ATP, GTP, CTPpseudo-UTP/5-methyl-CTP/ATP/GTP UTP-5-oxyacetic acid Me ester Xanthosine

According to the disclosure, polynucleotides of the disclosure may besynthesized to comprise the combinations or single modifications ofTable 1 or Table 2.

Where a single modification is listed, the listed nucleoside ornucleotide represents 100 percent of that A, U, G or C nucleotide ornucleoside having been modified. Where percentages are listed, theserepresent the percentage of that particular A, U, G or C nucleobasetriphosphate of the total amount of A, U, G, or C triphosphate present.For example, the combination: 25% 5-Aminoallyl-CTP+75% CTP/25%5-Methoxy-UTP+75% UTP refers to a polynucleotide where 25% of thecytosine triphosphates are 5-Aminoallyl-CTP while 75% of the cytosinesare CTP; whereas 25% of the uracils are 5-methoxy UTP while 75% of theuracils are UTP. Where no modified UTP is listed then the naturallyoccurring ATP, UTP, GTP and/or CTP is used at 100% of the sites of thosenucleotides found in the polynucleotide. In this example all of the GTPand ATP nucleotides are left unmodified.

The mRNAs of the present disclosure, or regions thereof, may be codonoptimized. Codon optimization methods are known in the art and may beuseful for a variety of purposes: matching codon frequencies in hostorganisms to ensure proper folding, bias GC content to increase mRNAstability or reduce secondary structures, minimize tandem repeat codonsor base runs that may impair gene construction or expression, customizetranscriptional and translational control regions, insert or removeproteins trafficking sequences, remove/add post translation modificationsites in encoded proteins (e.g., glycosylation sites), add, remove orshuffle protein domains, insert or delete restriction sites, modifyribosome binding sites and mRNA degradation sites, adjust translationrates to allow the various domains of the protein to fold properly, orto reduce or eliminate problem secondary structures within thepolynucleotide. Codon optimization tools, algorithms and services areknown in the art; non-limiting examples include services from GeneArt(Life Technologies), DNA2.0 (Menlo Park, Calif.) and/or proprietarymethods. In one embodiment, the mRNA sequence is optimized usingoptimization algorithms, e.g., to optimize expression in mammalian cellsor enhance mRNA stability.

In certain embodiments, the present disclosure includes polynucleotideshaving at least 80%, at least 85%, at least 90%, at least 95%, at least98%, or at least 99% sequence identity to any of the polynucleotidesequences described herein.

mRNAs of the present disclosure may be produced by means available inthe art, including but not limited to in vitro transcription (IVT) andsynthetic methods. Enzymatic (IVT), solid-phase, liquid-phase, combinedsynthetic methods, small region synthesis, and ligation methods may beutilized. In one embodiment, mRNAs are made using IVT enzymaticsynthesis methods. Methods of making polynucleotides by IVT are known inthe art and are described in International Application PCT/US2013/30062,the contents of which are incorporated herein by reference in theirentirety. Accordingly, the present disclosure also includespolynucleotides, e.g., DNA, constructs and vectors that may be used toin vitro transcribe an mRNA described herein.

Non-natural modified nucleobases may be introduced into polynucleotides,e.g., mRNA, during synthesis or post-synthesis. In certain embodiments,modifications may be on internucleoside linkages, purine or pyrimidinebases, or sugar. In particular embodiments, the modification may beintroduced at the terminal of a polynucleotide chain or anywhere else inthe polynucleotide chain; with chemical synthesis or with a polymeraseenzyme. Examples of modified nucleic acids and their synthesis aredisclosed in PCT application No. PCT/US2012/058519. Synthesis ofmodified polynucleotides is also described in Verma and Eckstein, AnnualReview of Biochemistry, vol. 76, 99-134 (1998).

Either enzymatic or chemical ligation methods may be used to conjugatepolynucleotides or their regions with different functional moieties,such as targeting or delivery agents, fluorescent labels, liquids,nanoparticles, etc. Conjugates of polynucleotides and modifiedpolynucleotides are reviewed in Goodchild, Bioconjugate Chemistry, vol.1(3), 165-187 (1990).

MicroRNA (miRNA) Binding Sites

Polynucleotides of the disclosure can include regulatory elements, forexample, microRNA (miRNA) binding sites, transcription factor bindingsites, structured mRNA sequences and/or motifs, artificial binding sitesengineered to act as pseudo-receptors for endogenous nucleic acidbinding molecules, and combinations thereof. In some embodiments,polynucleotides including such regulatory elements are referred to asincluding “sensor sequences.” Non-limiting examples of sensor sequencesare described in U.S. Publication 2014/0200261, the contents of whichare incorporated herein by reference in their entirety.

In some embodiments, a polynucleotide (e.g., a ribonucleic acid (RNA),e.g., a messenger RNA (mRNA)) of the disclosure comprises an openreading frame (ORF) encoding a polypeptide of interest and furthercomprises one or more miRNA binding site(s). Inclusion or incorporationof miRNA binding site(s) provides for regulation of polynucleotides ofthe disclosure, and in turn, of the polypeptides encoded therefrom,based on tissue-specific and/or cell-type specific expression ofnaturally-occurring miRNAs.

A miRNA, e.g., a natural-occurring miRNA, is a 19-25 nucleotide longnoncoding RNA that binds to a polynucleotide and down-regulates geneexpression either by reducing stability or by inhibiting translation ofthe polynucleotide. A miRNA sequence comprises a “seed” region, i.e., asequence in the region of positions 2-8 of the mature miRNA. A miRNAseed can comprise positions 2-8 or 2-7 of the mature miRNA. In someembodiments, a miRNA seed can comprise 7 nucleotides (e.g., nucleotides2-8 of the mature miRNA), wherein the seed-complementary site in thecorresponding miRNA binding site is flanked by an adenosine (A) opposedto miRNA position 1. In some embodiments, a miRNA seed can comprise 6nucleotides (e.g., nucleotides 2-7 of the mature miRNA), wherein theseed-complementary site in the corresponding miRNA binding site isflanked by an adenosine (A) opposed to miRNA position 1. See, forexample, Grimson A, Farh K K, Johnston W K, Garrett-Engele P, Lim L P,Bartel D P; Mol Cell. 2007 Jul. 6; 27(1):91-105. miRNA profiling of thetarget cells or tissues can be conducted to determine the presence orabsence of miRNA in the cells or tissues. In some embodiments, apolynucleotide (e.g., a ribonucleic acid (RNA), e.g., a messenger RNA(mRNA)) of the disclosure comprises one or more microRNA binding sites,microRNA target sequences, microRNA complementary sequences, or microRNAseed complementary sequences. Such sequences can correspond to, e.g.,have complementarity to, any known microRNA such as those taught in USPublication US2005/0261218 and US Publication US2005/0059005, thecontents of each of which are incorporated herein by reference in theirentirety.

As used herein, the term “microRNA (miRNA or miR) binding site” refersto a sequence within a polynucleotide, e.g., within a DNA or within anRNA transcript, including in the 5′UTR and/or 3′UTR, that has sufficientcomplementarity to all or a region of a miRNA to interact with,associate with or bind to the miRNA. In some embodiments, apolynucleotide of the disclosure comprising an ORF encoding apolypeptide of interest and further comprises one or more miRNA bindingsite(s). In exemplary embodiments, a 5′UTR and/or 3′UTR of thepolynucleotide (e.g., a ribonucleic acid (RNA), e.g., a messenger RNA(mRNA)) comprises the one or more miRNA binding site(s).

A miRNA binding site having sufficient complementarity to a miRNA refersto a degree of complementarity sufficient to facilitate miRNA-mediatedregulation of a polynucleotide, e.g., miRNA-mediated translationalrepression or degradation of the polynucleotide. In exemplary aspects ofthe disclosure, a miRNA binding site having sufficient complementarityto the miRNA refers to a degree of complementarity sufficient tofacilitate miRNA-mediated degradation of the polynucleotide, e.g.,miRNA-guided RNA-induced silencing complex (RISC)-mediated cleavage ofmRNA. The miRNA binding site can have complementarity to, for example, a19-25 nucleotide miRNA sequence, to a 19-23 nucleotide miRNA sequence,or to a 22 nucleotide miRNA sequence. A miRNA binding site can becomplementary to only a portion of a miRNA, e.g., to a portion less than1, 2, 3, or 4 nucleotides of the full length of a naturally-occurringmiRNA sequence. Full or complete complementarity (e.g., fullcomplementarity or complete complementarity over all or a significantportion of the length of a naturally-occurring miRNA) is preferred whenthe desired regulation is mRNA degradation.

In some embodiments, a miRNA binding site includes a sequence that hascomplementarity (e.g., partial or complete complementarity) with a miRNAseed sequence. In some embodiments, the miRNA binding site includes asequence that has complete complementarity with a miRNA seed sequence.In some embodiments, a miRNA binding site includes a sequence that hascomplementarity (e.g., partial or complete complementarity) with anmiRNA sequence. In some embodiments, the miRNA binding site includes asequence that has complete complementarity with a miRNA sequence. Insome embodiments, a miRNA binding site has complete complementarity witha miRNA sequence but for 1, 2, or 3 nucleotide substitutions, terminaladditions, and/or truncations.

In some embodiments, the miRNA binding site is the same length as thecorresponding miRNA. In other embodiments, the miRNA binding site isone, two, three, four, five, six, seven, eight, nine, ten, eleven ortwelve nucleotide(s) shorter than the corresponding miRNA at the 5′terminus, the 3′ terminus, or both. In still other embodiments, themicroRNA binding site is two nucleotides shorter than the correspondingmicroRNA at the 5′ terminus, the 3′ terminus, or both. The miRNA bindingsites that are shorter than the corresponding miRNAs are still capableof degrading the mRNA incorporating one or more of the miRNA bindingsites or preventing the mRNA from translation.

In some embodiments, the miRNA binding site binds the correspondingmature miRNA that is part of an active RISC containing Dicer. In anotherembodiment, binding of the miRNA binding site to the corresponding miRNAin RISC degrades the mRNA containing the miRNA binding site or preventsthe mRNA from being translated. In some embodiments, the miRNA bindingsite has sufficient complementarity to miRNA so that a RISC complexcomprising the miRNA cleaves the polynucleotide comprising the miRNAbinding site. In other embodiments, the miRNA binding site has imperfectcomplementarity so that a RISC complex comprising the miRNA inducesinstability in the polynucleotide comprising the miRNA binding site. Inanother embodiment, the miRNA binding site has imperfect complementarityso that a RISC complex comprising the miRNA represses transcription ofthe polynucleotide comprising the miRNA binding site.

In some embodiments, the miRNA binding site has one, two, three, four,five, six, seven, eight, nine, ten, eleven or twelve mismatch(es) fromthe corresponding miRNA.

In some embodiments, the miRNA binding site has at least about ten, atleast about eleven, at least about twelve, at least about thirteen, atleast about fourteen, at least about fifteen, at least about sixteen, atleast about seventeen, at least about eighteen, at least about nineteen,at least about twenty, or at least about twenty-one contiguousnucleotides complementary to at least about ten, at least about eleven,at least about twelve, at least about thirteen, at least about fourteen,at least about fifteen, at least about sixteen, at least aboutseventeen, at least about eighteen, at least about nineteen, at leastabout twenty, or at least about twenty-one, respectively, contiguousnucleotides of the corresponding miRNA.

By engineering one or more miRNA binding sites into a polynucleotide ofthe disclosure, the polynucleotide can be targeted for degradation orreduced translation, provided the miRNA in question is available. Thiscan reduce off-target effects upon delivery of the polynucleotide. Forexample, if a polynucleotide of the disclosure is not intended to bedelivered to a tissue or cell but ends up is said tissue or cell, then amiRNA abundant in the tissue or cell can inhibit the expression of thegene of interest if one or multiple binding sites of the miRNA areengineered into the 5′UTR and/or 3′UTR of the polynucleotide.

Conversely, miRNA binding sites can be removed from polynucleotidesequences in which they naturally occur in order to increase proteinexpression in specific tissues. For example, a binding site for aspecific miRNA can be removed from a polynucleotide to improve proteinexpression in tissues or cells containing the miRNA.

In one embodiment, a polynucleotide of the disclosure can include atleast one miRNA-binding site in the 5′UTR and/or 3′UTR in order toregulate cytotoxic or cytoprotective mRNA therapeutics to specific cellssuch as, but not limited to, normal and/or cancerous cells. In anotherembodiment, a polynucleotide of the disclosure can include two, three,four, five, six, seven, eight, nine, ten, or more miRNA-binding sites inthe 5′-UTR and/or 3′-UTR in order to regulate cytotoxic orcytoprotective mRNA therapeutics to specific cells such as, but notlimited to, normal and/or cancerous cells.

Regulation of expression in multiple tissues can be accomplished throughintroduction or removal of one or more miRNA binding sites, e.g., one ormore distinct miRNA binding sites. The decision whether to remove orinsert a miRNA binding site can be made based on miRNA expressionpatterns and/or their profilings in tissues and/or cells in developmentand/or disease. Identification of miRNAs, miRNA binding sites, and theirexpression patterns and role in biology have been reported (e.g.,Bonauer et al., Curr Drug Targets 2010 11:943-949; Anand and ChereshCurr Opin Hematol 2011 18:171-176; Contreras and Rao Leukemia 201226:404-413 (2011 Dec. 20. doi: 10.1038/leu.2011.356); Bartel Cell 2009136:215-233; Landgraf et al, Cell, 2007 129:1401-1414; Gentner andNaldini, Tissue Antigens. 2012 80:393-403 and all references therein;each of which is incorporated herein by reference in its entirety).

miRNAs and miRNA binding sites can correspond to any known sequence,including non-limiting examples described in U.S. Publication Nos.2014/0200261, 2005/0261218, and 2005/0059005, each of which areincorporated herein by reference in their entirety.

Examples of tissues where miRNA are known to regulate mRNA, and therebyprotein expression, include, but are not limited to, liver (miR-122),muscle (miR-133, miR-206, miR-208), endothelial cells (miR-17-92,miR-126), myeloid cells (miR-142-3p, miR-142-5p, miR-16, miR-21,miR-223, miR-24, miR-27), adipose tissue (let-7, miR-30c), heart(miR-1d, miR-149), kidney (miR-192, miR-194, miR-204), and lungepithelial cells (let-7, miR-133, miR-126).

Specifically, miRNAs are known to be differentially expressed in immunecells (also called hematopoietic cells), such as antigen presentingcells (APCs) (e.g., dendritic cells and macrophages), macrophages,monocytes, B lymphocytes, T lymphocytes, granulocytes, natural killercells, etc. Immune cell specific miRNAs are involved in immunogenicity,autoimmunity, the immune response to infection, inflammation, as well asunwanted immune response after gene therapy and tissue/organtransplantation. Immune cell specific miRNAs also regulate many aspectsof development, proliferation, differentiation and apoptosis ofhematopoietic cells (immune cells). For example, miR-142 and miR-146 areexclusively expressed in immune cells, particularly abundant in myeloiddendritic cells. It has been demonstrated that the immune response to apolynucleotide can be shut-off by adding miR-142 binding sites to the3′-UTR of the polynucleotide, enabling more stable gene transfer intissues and cells. miR-142 efficiently degrades exogenouspolynucleotides in antigen presenting cells and suppresses cytotoxicelimination of transduced cells (e.g., Annoni A et al., blood, 2009,114, 5152-5161; Brown B D, et al., Nat med. 2006, 12(5), 585-591; BrownB D, et al., blood, 2007, 110(13): 4144-4152, each of which isincorporated herein by reference in its entirety).

An antigen-mediated immune response can refer to an immune responsetriggered by foreign antigens, which, when entering an organism, areprocessed by the antigen presenting cells and displayed on the surfaceof the antigen presenting cells. T cells can recognize the presentedantigen and induce a cytotoxic elimination of cells that express theantigen.

Introducing a miR-142 binding site into the 5′UTR and/or 3′UTR of apolynucleotide of the disclosure can selectively repress gene expressionin antigen presenting cells through miR-142 mediated degradation,limiting antigen presentation in antigen presenting cells (e.g.,dendritic cells) and thereby preventing antigen-mediated immune responseafter the delivery of the polynucleotide. The polynucleotide is thenstably expressed in target tissues or cells without triggering cytotoxicelimination.

In one embodiment, binding sites for miRNAs that are known to beexpressed in immune cells, in particular, antigen presenting cells, canbe engineered into a polynucleotide of the disclosure to suppress theexpression of the polynucleotide in antigen presenting cells throughmiRNA mediated RNA degradation, subduing the antigen-mediated immuneresponse. Expression of the polynucleotide is maintained in non-immunecells where the immune cell specific miRNAs are not expressed. Forexample, in some embodiments, to prevent an immunogenic reaction againsta liver specific protein, any miR-122 binding site can be removed and amiR-142 (and/or mirR-146) binding site can be engineered into the 5′UTRand/or 3′UTR of a polynucleotide of the disclosure.

To further drive the selective degradation and suppression in APCs andmacrophage, a polynucleotide of the disclosure can include a furthernegative regulatory element in the 5′UTR and/or 3′UTR, either alone orin combination with miR-142 and/or miR-146 binding sites. As anon-limiting example, the further negative regulatory element is aConstitutive Decay Element (CDE).

Immune cell specific miRNAs include, but are not limited to,hsa-let-7a-2-3p, hsa-let-7a-3p, hsa-7a-5p, hsa-let-7c, hsa-let-7e-3p,hsa-let-7e-5p, hsa-let-7g-3p, hsa-let-7g-5p, hsa-let-7i-3p,hsa-let-7i-5p, miR-10a-3p, miR-10a-5p, miR-1184, hsa-let-7f-1-3p,hsa-let-7f-2-5p, hsa-let-7f-5p, miR-125b-1-3p, miR-125b-2-3p,miR-125b-5p, miR-1279, miR-130a-3p, miR-130a-5p, miR-132-3p, miR-132-5p,miR-142-3p, miR-142-5p, miR-143-3p, miR-143-5p, miR-146a-3p,miR-146a-5p, miR-146b-3p, miR-146b-5p, miR-147a, miR-147b, miR-148a-5p,miR-148a-3p, miR-150-3p, miR-150-5p, miR-151b, miR-155-3p, miR-155-5p,miR-15a-3p, miR-15a-5p, miR-15b-5p, miR-15b-3p, miR-16-1-3p,miR-16-2-3p, miR-16-5p, miR-17-5p, miR-181a-3p, miR-181a-5p,miR-181a-2-3p, miR-182-3p, miR-182-5p, miR-197-3p, miR-197-5p,miR-21-5p, miR-21-3p, miR-214-3p, miR-214-5p, miR-223-3p, miR-223-5p,miR-221-3p, miR-221-5p, miR-23b-3p, miR-23b-5p, miR-24-1-5p,miR-24-2-5p, miR-24-3p, miR-26a-1-3p, miR-26a-2-3p, miR-26a-5p,miR-26b-3p, miR-26b-5p, miR-27a-3p, miR-27a-5p, miR-27b-3p, miR-27b-5p,miR-28-3p, miR-28-5p, miR-2909, miR-29a-3p, miR-29a-5p, miR-29b-1-5p,miR-29b-2-5p, miR-29c-3p, miR-29c-5p, miR-30e-3p, miR-30e-5p,miR-331-5p, miR-339-3p, miR-339-5p, miR-345-3p, miR-345-5p, miR-346,miR-34a-3p, miR-34a-5p, miR-363-3p, miR-363-5p, miR-372, miR-377-3p,miR-377-5p, miR-493-3p, miR-493-5p, miR-542, miR-548b-5p, miR548c-5p,miR-548i, miR-548j, miR-548n, miR-574-3p, miR-598, miR-718, miR-935,miR-99a-3p, miR-99a-5p, miR-99b-3p, and miR-99b-5p. Furthermore, novelmiRNAs can be identified in immune cell through micro-arrayhybridization and microtome analysis (e.g., Jima D D et al, Blood, 2010,116:e118-e127; Vaz C et al., BMC Genomics, 2010, 11, 288, the content ofeach of which is incorporated herein by reference in its entirety.)

miRNAs that are known to be expressed in the liver include, but are notlimited to, miR-107, miR-122-3p, miR-122-5p, miR-1228-3p, miR-1228-5p,miR-1249, miR-129-5p, miR-1303, miR-151a-3p, miR-151a-5p, miR-152,miR-194-3p, miR-194-5p, miR-199a-3p, miR-199a-5p, miR-199b-3p,miR-199b-5p, miR-296-5p, miR-557, miR-581, miR-939-3p, and miR-939-5p.miRNA binding sites from any liver specific miRNA can be introduced toor removed from a polynucleotide of the disclosure to regulateexpression of the polynucleotide in the liver. Liver specific miRNAbinding sites can be engineered alone or further in combination withimmune cell (e.g., APC) miRNA binding sites in a polynucleotide of thedisclosure.

miRNAs that are known to be expressed in the lung include, but are notlimited to, let-7a-2-3p, let-7a-3p, let-7a-5p, miR-126-3p, miR-126-5p,miR-127-3p, miR-127-5p, miR-130a-3p, miR-130a-5p, miR-130b-3p,miR-130b-5p, miR-133a, miR-133b, miR-134, miR-18a-3p, miR-18a-5p,miR-18b-3p, miR-18b-5p, miR-24-1-5p, miR-24-2-5p, miR-24-3p, miR-296-3p,miR-296-5p, miR-32-3p, miR-337-3p, miR-337-5p, miR-381-3p, andmiR-381-5p. miRNA binding sites from any lung specific miRNA can beintroduced to or removed from a polynucleotide of the disclosure toregulate expression of the polynucleotide in the lung. Lung specificmiRNA binding sites can be engineered alone or further in combinationwith immune cell (e.g., APC) miRNA binding sites in a polynucleotide ofthe disclosure.

miRNAs that are known to be expressed in the heart include, but are notlimited to, miR-1, miR-133a, miR-133b, miR-149-3p, miR-149-5p,miR-186-3p, miR-186-5p, miR-208a, miR-208b, miR-210, miR-296-3p,miR-320, miR-451a, miR-451b, miR-499a-3p, miR-499a-5p, miR-499b-3p,miR-499b-5p, miR-744-3p, miR-744-5p, miR-92b-3p, and miR-92b-5p. miRNAbinding sites from any heart specific microRNA can be introduced to orremoved from a polynucleotide of the disclosure to regulate expressionof the polynucleotide in the heart. Heart specific miRNA binding sitescan be engineered alone or further in combination with immune cell(e.g., APC) miRNA binding sites in a polynucleotide of the disclosure.

miRNAs that are known to be expressed in the nervous system include, butare not limited to, miR-124-5p, miR-125a-3p, miR-125a-5p, miR-125b-1-3p,miR-125b-2-3p, miR-125b-5p, miR-1271-3p, miR-1271-5p, miR-128,miR-132-5p, miR-135a-3p, miR-135a-5p, miR-135b-3p, miR-135b-5p, miR-137,miR-139-5p, miR-139-3p, miR-149-3p, miR-149-5p, miR-153, miR-181c-3p,miR-181c-5p, miR-183-3p, miR-183-5p, miR-190a, miR-190b, miR-212-3p,miR-212-5p, miR-219-1-3p, miR-219-2-3p, miR-23a-3p, miR-23a-5p,miR-30a-5p, miR-30b-3p, miR-30b-5p, miR-30c-1-3p, miR-30c-2-3p,miR-30c-5p, miR-30d-3p, miR-30d-5p, miR-329, miR-342-3p, miR-3665,miR-3666, miR-380-3p, miR-380-5p, miR-383, miR-410, miR-425-3p,miR-425-5p, miR-454-3p, miR-454-5p, miR-483, miR-510, miR-516a-3p,miR-548b-5p, miR-548c-5p, miR-571, miR-7-1-3p, miR-7-2-3p, miR-7-5p,miR-802, miR-922, miR-9-3p, and miR-9-5p. miRNAs enriched in the nervoussystem further include those specifically expressed in neurons,including, but not limited to, miR-132-3p, miR-132-3p, miR-148b-3p,miR-148b-5p, miR-151a-3p, miR-151a-5p, miR-212-3p, miR-212-5p, miR-320b,miR-320e, miR-323a-3p, miR-323a-5p, miR-324-5p, miR-325, miR-326,miR-328, miR-922 and those specifically expressed in glial cells,including, but not limited to, miR-1250, miR-219-1-3p, miR-219-2-3p,miR-219-5p, miR-23a-3p, miR-23a-5p, miR-3065-3p, miR-3065-5p,miR-30e-3p, miR-30e-5p, miR-32-5p, miR-338-5p, and miR-657. miRNAbinding sites from any CNS specific miRNA can be introduced to orremoved from a polynucleotide of the disclosure to regulate expressionof the polynucleotide in the nervous system. Nervous system specificmiRNA binding sites can be engineered alone or further in combinationwith immune cell (e.g., APC) miRNA binding sites in a polynucleotide ofthe disclosure.

miRNAs that are known to be expressed in the pancreas include, but arenot limited to, miR-105-3p, miR-105-5p, miR-184, miR-195-3p, miR-195-5p,miR-196a-3p, miR-196a-5p, miR-214-3p, miR-214-5p, miR-216a-3p,miR-216a-5p, miR-30a-3p, miR-33a-3p, miR-33a-5p, miR-375, miR-7-1-3p,miR-7-2-3p, miR-493-3p, miR-493-5p, and miR-944. miRNA binding sitesfrom any pancreas specific miRNA can be introduced to or removed from apolynucleotide of the disclosure to regulate expression of thepolynucleotide in the pancreas. Pancreas specific miRNA binding sitescan be engineered alone or further in combination with immune cell (e.g.APC) miRNA binding sites in a polynucleotide of the disclosure.

miRNAs that are known to be expressed in the kidney include, but are notlimited to, miR-122-3p, miR-145-5p, miR-17-5p, miR-192-3p, miR-192-5p,miR-194-3p, miR-194-5p, miR-20a-3p, miR-20a-5p, miR-204-3p, miR-204-5p,miR-210, miR-216a-3p, miR-216a-5p, miR-296-3p, miR-30a-3p, miR-30a-5p,miR-30b-3p, miR-30b-5p, miR-30c-1-3p, miR-30c-2-3p, miR30c-5p,miR-324-3p, miR-335-3p, miR-335-5p, miR-363-3p, miR-363-5p, and miR-562.miRNA binding sites from any kidney specific miRNA can be introduced toor removed from a polynucleotide of the disclosure to regulateexpression of the polynucleotide in the kidney. Kidney specific miRNAbinding sites can be engineered alone or further in combination withimmune cell (e.g., APC) miRNA binding sites in a polynucleotide of thedisclosure.

miRNAs that are known to be expressed in the muscle include, but are notlimited to, let-7g-3p, let-7g-5p, miR-1, miR-1286, miR-133a, miR-133b,miR-140-3p, miR-143-3p, miR-143-5p, miR-145-3p, miR-145-5p, miR-188-3p,miR-188-5p, miR-206, miR-208a, miR-208b, miR-25-3p, and miR-25-5p. miRNAbinding sites from any muscle specific miRNA can be introduced to orremoved from a polynucleotide of the disclosure to regulate expressionof the polynucleotide in the muscle. Muscle specific miRNA binding sitescan be engineered alone or further in combination with immune cell(e.g., APC) miRNA binding sites in a polynucleotide of the disclosure.

miRNAs are also differentially expressed in different types of cells,such as, but not limited to, endothelial cells, epithelial cells, andadipocytes.

miRNAs that are known to be expressed in endothelial cells include, butare not limited to, let-7b-3p, let-7b-5p, miR-100-3p, miR-100-5p,miR-101-3p, miR-101-5p, miR-126-3p, miR-126-5p, miR-1236-3p,miR-1236-5p, miR-130a-3p, miR-130a-5p, miR-17-5p, miR-17-3p, miR-18a-3p,miR-18a-5p, miR-19a-3p, miR-19a-5p, miR-19b-1-5p, miR-19b-2-5p,miR-19b-3p, miR-20a-3p, miR-20a-5p, miR-217, miR-210, miR-21-3p,miR-21-5p, miR-221-3p, miR-221-5p, miR-222-3p, miR-222-5p, miR-23a-3p,miR-23a-5p, miR-296-5p, miR-361-3p, miR-361-5p, miR-421, miR-424-3p,miR-424-5p, miR-513a-5p, miR-92a-1-5p, miR-92a-2-5p, miR-92a-3p,miR-92b-3p, and miR-92b-5p. Many novel miRNAs are discovered inendothelial cells from deep-sequencing analysis (e.g., Voellenkle C etal., RNA, 2012, 18, 472-484, incorporated herein by reference in itsentirety). miRNA binding sites from any endothelial cell specific miRNAcan be introduced to or removed from a polynucleotide of the disclosureto regulate expression of the polynucleotide in the endothelial cells.

miRNAs that are known to be expressed in epithelial cells include, butare not limited to, let-7b-3p, let-7b-5p, miR-1246, miR-200a-3p,miR-200a-5p, miR-200b-3p, miR-200b-5p, miR-200c-3p, miR-200c-5p,miR-338-3p, miR-429, miR-451a, miR-451b, miR-494, miR-802 and miR-34a,miR-34b-5p, miR-34c-5p, miR-449a, miR-449b-3p, miR-449b-5p specific inrespiratory ciliated epithelial cells, let-7 family, miR-133a, miR-133b,miR-126 specific in lung epithelial cells, miR-382-3p, miR-382-5pspecific in renal epithelial cells, and miR-762 specific in cornealepithelial cells. miRNA binding sites from any epithelial cell specificmiRNA can be introduced to or removed from a polynucleotide of thedisclosure to regulate expression of the polynucleotide in theepithelial cells.

In addition, a large group of miRNAs are enriched in embryonic stemcells, controlling stem cell self-renewal as well as the developmentand/or differentiation of various cell lineages, such as neural cells,cardiac, hematopoietic cells, skin cells, osteogenic cells and musclecells (e.g., Kuppusamy K T et al., Curr. Mol Med, 2013, 13(5), 757-764;Vidigal J A and Ventura A, Semin Cancer Biol. 2012, 22(5-6), 428-436;Goff L A et al., PLoS One, 2009, 4:e7192; Morin R D et al., Genome Res,2008, 18, 610-621; Yoo J K et al., Stem Cells Dev. 2012, 21(11),2049-2057, each of which is incorporated herein by reference in itsentirety). miRNAs abundant in embryonic stem cells include, but are notlimited to, let-7a-2-3p, let-a-3p, let-7a-5p, let7d-3p, let-7d-5p,miR-103a-2-3p, miR-103a-5p, miR-106b-3p, miR-106b-5p, miR-1246,miR-1275, miR-138-1-3p, miR-138-2-3p, miR-138-5p, miR-154-3p,miR-154-5p, miR-200c-3p, miR-200c-5p, miR-290, miR-301a-3p, miR-301a-5p,miR-302a-3p, miR-302a-5p, miR-302b-3p, miR-302b-5p, miR-302c-3p,miR-302c-5p, miR-302d-3p, miR-302d-5p, miR-302e, miR-367-3p, miR-367-5p,miR-369-3p, miR-369-5p, miR-370, miR-371, miR-373, miR-380-5p,miR-423-3p, miR-423-5p, miR-486-5p, miR-520c-3p, miR-548e, miR-548f,miR-548g-3p, miR-548g-5p, miR-548i, miR-548k, miR-5481, miR-548m,miR-548n, miR-548o-3p, miR-548o-5p, miR-548p, miR-664a-3p, miR-664a-5p,miR-664b-3p, miR-664b-5p, miR-766-3p, miR-766-5p, miR-885-3p,miR-885-5p, miR-93-3p, miR-93-5p, miR-941, miR-96-3p, miR-96-5p,miR-99b-3p and miR-99b-5p. Many predicted novel miRNAs are discovered bydeep sequencing in human embryonic stem cells (e.g., Morin R D et al.,Genome Res, 2008, 18, 610-621; Goff L A et al., PLoS One, 2009, 4:e7192;Bar M et al., Stem cells, 2008, 26, 2496-2505, the content of each ofwhich is incorporated herein by reference in its entirety).

In one embodiment, the binding sites of embryonic stem cell specificmiRNAs can be included in or removed from the 3′UTR of a polynucleotideof the disclosure to modulate the development and/or differentiation ofembryonic stem cells, to inhibit the senescence of stem cells in adegenerative condition (e.g. degenerative diseases), or to stimulate thesenescence and apoptosis of stem cells in a disease condition (e.g.cancer stem cells).

Many miRNA expression studies are conducted to profile the differentialexpression of miRNAs in various cancer cells/tissues and other diseases.Some miRNAs are abnormally over-expressed in certain cancer cells andothers are under-expressed. For example, miRNAs are differentiallyexpressed in cancer cells (WO2008/154098, US2013/0059015,US2013/0042333, WO2011/157294); cancer stem cells (US2012/0053224);pancreatic cancers and diseases (US2009/0131348, US2011/0171646,US2010/0286232, U.S. Pat. No. 8,389,210); asthma and inflammation (U.S.Pat. No. 8,415,096); prostate cancer (US2013/0053264); hepatocellularcarcinoma (WO2012/151212, US2012/0329672, WO2008/054828, U.S. Pat. No.8,252,538); lung cancer cells (WO2011/076143, WO2013/033640,WO2009/070653, US2010/0323357); cutaneous T cell lymphoma(WO2013/011378); colorectal cancer cells (WO2011/0281756,WO2011/076142); cancer positive lymph nodes (WO2009/100430,US2009/0263803); nasopharyngeal carcinoma (EP2112235); chronicobstructive pulmonary disease (US2012/0264626, US2013/0053263); thyroidcancer (WO2013/066678); ovarian cancer cells (US2012/0309645,WO2011/095623); breast cancer cells (WO2008/154098, WO2007/081740,US2012/0214699), leukemia and lymphoma (WO2008/073915, US2009/0092974,US2012/0316081, US2012/0283310, WO2010/018563), the content of each ofwhich is incorporated herein by reference in its entirety.

As a non-limiting example, miRNA binding sites for miRNAs that areover-expressed in certain cancer and/or tumor cells can be removed fromthe 3′UTR of a polynucleotide of the disclosure, restoring theexpression suppressed by the over-expressed miRNAs in cancer cells, thusameliorating the corresponsive biological function, for instance,transcription stimulation and/or repression, cell cycle arrest,apoptosis and cell death. Normal cells and tissues, wherein miRNAsexpression is not up-regulated, will remain unaffected.

miRNA can also regulate complex biological processes such asangiogenesis (e.g., miR-132) (Anand and Cheresh Curr Opin Hematol 201118:171-176). In the polynucleotides of the disclosure, miRNA bindingsites that are involved in such processes can be removed or introduced,in order to tailor the expression of the polynucleotides to biologicallyrelevant cell types or relevant biological processes. In this context,the polynucleotides of the disclosure are defined as auxotrophicpolynucleotides.

In some embodiments, the therapeutic window and/or differentialexpression (e.g., tissue-specific expression) of a polypeptide of thedisclosure may be altered by incorporation of a miRNA binding site intoan mRNA encoding the polypeptide. In one example, an mRNA may includeone or more miRNA binding sites that are bound by miRNAs that havehigher expression in one tissue type as compared to another. In anotherexample, an mRNA may include one or more miRNA binding sites that arebound by miRNAs that have lower expression in a cancer cell as comparedto a non-cancerous cell of the same tissue of origin. When present in acancer cell that expresses low levels of such an miRNA, the polypeptideencoded by the mRNA typically will show increased expression.

Liver cancer cells (e.g., hepatocellular carcinoma cells) typicallyexpress low levels of miR-122 as compared to normal liver cells.Therefore, an mRNA encoding a polypeptide that includes at least onemiR-122 binding site (e.g., in the 3′-UTR of the mRNA) will typicallyexpress comparatively low levels of the polypeptide in normal livercells and comparatively high levels of the polypeptide in liver cancercells.

In some embodiments, the mRNA includes at least one miR-122 bindingsite, at least two miR-122 binding sites, at least three miR-122 bindingsites, at least four miR-122 binding sites, or at least five miR-122binding sites. In one aspect, the miRNA binding site binds miR-122 or iscomplementary to miR-122. In another aspect, the miRNA binding sitebinds to miR-122-3p or miR-122-5p. In a particular aspect, the miRNAbinding site comprises a nucleotide sequence at least 80%, at least 85%,at least 90%, at least 95%, or 100% identical to SEQ ID NO: 175, whereinthe miRNA binding site binds to miR-122. In another particular aspect,the miRNA binding site comprises a nucleotide sequence at least 80%, atleast 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:173, wherein the miRNA binding site binds to miR-122. These sequencesare shown below in Table 3.

In some embodiments, a polynucleotide of the disclosure comprises amiRNA binding site, wherein the miRNA binding site comprises one or morenucleotide sequences selected from Table 3, including one or more copiesof any one or more of the miRNA binding site sequences. In someembodiments, a polynucleotide of the disclosure further comprises atleast one, two, three, four, five, six, seven, eight, nine, ten, or moreof the same or different miRNA binding sites selected from Table 3,including any combination thereof. In some embodiments, the miRNAbinding site binds to miR-142 or is complementary to miR-142. In someembodiments, the miR-142 comprises SEQ ID NO: 27. In some embodiments,the miRNA binding site binds to miR-142-3p or miR-142-5p. In someembodiments, the miR-142-3p binding site comprises SEQ ID NO: 29. Insome embodiments, the miR-142-5p binding site comprises SEQ ID NO: 31.In some embodiments, the miRNA binding site comprises a nucleotidesequence at least 80%, at least 85%, at least 90%, at least 95%, or 100%identical to SEQ ID NO: 29 or SEQ ID NO: 31

TABLE 3 Representative microRNAs and microRNA binding sites SEQ ID NO.Description Sequence 27 miR-142 GACAGUGCAGUCACCCAUAAAGUAGAAAGCACUACUAACAGCACUGGAGGGUGUAGUGUUUCC UACUUUAUGGAUGAGUGUACUGUG 28 miR-142-3pUGUAGUGUUUCCUACUUUAUGGA 29 miR-142-3p UCCAUAAAGUAGGAAACACUACA bindingsite 30 miR-142-5p CAUAAAGUAGAAAGCACUACU 31 miR-142-5pAGUAGUGCUUUCUACUUUAUG binding site 171 miR-122CCUUAGCAGAGCUGUGGAGUGUGACAAUGGU GUUUGUGUCUAAACUAUCAAACGCCAUUAUCACACUAAAUAGCUACUGCUAGGC 172 miR-122-3p AACGCCAUUAUCACACUAAAUA 173miR-122-3p UAUUUAGUGUGAUAAUGGCGUU binding site 174 miR-122-5pUGGAGUGUGACAAUGGUGUUUG 175 miR-122-5p CAAACACCAUUGUCACACUCCA bindingsite

In some embodiments, a miRNA binding site is inserted in thepolynucleotide of the disclosure in any position of the polynucleotide(e.g., the 5′UTR and/or 3′UTR). In some embodiments, the 5′UTR comprisesa miRNA binding site. In some embodiments, the 3′UTR comprises a miRNAbinding site. In some embodiments, the 5′UTR and the 3′UTR comprise amiRNA binding site. The insertion site in the polynucleotide can beanywhere in the polynucleotide as long as the insertion of the miRNAbinding site in the polynucleotide does not interfere with thetranslation of a functional polypeptide in the absence of thecorresponding miRNA; and in the presence of the miRNA, the insertion ofthe miRNA binding site in the polynucleotide and the binding of themiRNA binding site to the corresponding miRNA are capable of degradingthe polynucleotide or preventing the translation of the polynucleotide.

In some embodiments, a miRNA binding site is inserted in at least about30 nucleotides downstream from the stop codon of an ORF in apolynucleotide of the disclosure comprising the ORF. In someembodiments, a miRNA binding site is inserted in at least about 10nucleotides, at least about 15 nucleotides, at least about 20nucleotides, at least about 25 nucleotides, at least about 30nucleotides, at least about 35 nucleotides, at least about 40nucleotides, at least about 45 nucleotides, at least about 50nucleotides, at least about 55 nucleotides, at least about 60nucleotides, at least about 65 nucleotides, at least about 70nucleotides, at least about 75 nucleotides, at least about 80nucleotides, at least about 85 nucleotides, at least about 90nucleotides, at least about 95 nucleotides, or at least about 100nucleotides downstream from the stop codon of an ORF in a polynucleotideof the disclosure. In some embodiments, a miRNA binding site is insertedin about 10 nucleotides to about 100 nucleotides, about 20 nucleotidesto about 90 nucleotides, about 30 nucleotides to about 80 nucleotides,about 40 nucleotides to about 70 nucleotides, about 50 nucleotides toabout 60 nucleotides, about 45 nucleotides to about 65 nucleotidesdownstream from the stop codon of an ORF in a polynucleotide of thedisclosure.

miRNA gene regulation can be influenced by the sequence surrounding themiRNA such as, but not limited to, the species of the surroundingsequence, the type of sequence (e.g., heterologous, homologous,exogenous, endogenous, or artificial), regulatory elements in thesurrounding sequence and/or structural elements in the surroundingsequence. The miRNA can be influenced by the 5′UTR and/or 3′UTR. As anon-limiting example, a non-human 3′UTR can increase the regulatoryeffect of the miRNA sequence on the expression of a polypeptide ofinterest compared to a human 3′UTR of the same sequence type.

In one embodiment, other regulatory elements and/or structural elementsof the 5′UTR can influence miRNA mediated gene regulation. One exampleof a regulatory element and/or structural element is a structured IRES(Internal Ribosome Entry Site) in the 5′UTR, which is necessary for thebinding of translational elongation factors to initiate proteintranslation. EIF4A2 binding to this secondarily structured element inthe 5′-UTR is necessary for miRNA mediated gene expression (Meijer H Aet al., Science, 2013, 340, 82-85, incorporated herein by reference inits entirety). The polynucleotides of the disclosure can further includethis structured 5′UTR in order to enhance microRNA mediated generegulation.

At least one miRNA binding site can be engineered into the 3′UTR of apolynucleotide of the disclosure. In this context, at least two, atleast three, at least four, at least five, at least six, at least seven,at least eight, at least nine, at least ten, or more miRNA binding sitescan be engineered into a 3′UTR of a polynucleotide of the disclosure.For example, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1to 3, 2, or 1 miRNA binding sites can be engineered into the 3′UTR of apolynucleotide of the disclosure. In one embodiment, miRNA binding sitesincorporated into a polynucleotide of the disclosure can be the same orcan be different miRNA sites. A combination of different miRNA bindingsites incorporated into a polynucleotide of the disclosure can includecombinations in which more than one copy of any of the different miRNAsites are incorporated. In another embodiment, miRNA binding sitesincorporated into a polynucleotide of the disclosure can target the sameor different tissues in the body. As a non-limiting example, through theintroduction of tissue-, cell-type-, or disease-specific miRNA bindingsites in the 3′-UTR of a polynucleotide of the disclosure, the degree ofexpression in specific cell types (e.g., hepatocytes, myeloid cells,endothelial cells, cancer cells, etc.) can be reduced.

In one embodiment, a miRNA binding site can be engineered near the 5′terminus of the 3′UTR, about halfway between the 5′ terminus and 3′terminus of the 3′UTR and/or near the 3′ terminus of the 3′UTR in apolynucleotide of the disclosure. As a non-limiting example, a miRNAbinding site can be engineered near the 5′ terminus of the 3′UTR andabout halfway between the 5′ terminus and 3′ terminus of the 3′UTR. Asanother non-limiting example, a miRNA binding site can be engineerednear the 3′ terminus of the 3′UTR and about halfway between the 5′terminus and 3′ terminus of the 3′UTR. As yet another non-limitingexample, a miRNA binding site can be engineered near the 5′ terminus ofthe 3′UTR and near the 3′ terminus of the 3′UTR.

In another embodiment, a 3′UTR can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10 miRNA binding sites. The miRNA binding sites can be complementaryto a miRNA, miRNA seed sequence, and/or miRNA sequences flanking theseed sequence.

In one embodiment, a polynucleotide of the disclosure can be engineeredto include more than one miRNA site expressed in different tissues ordifferent cell types of a subject. As a non-limiting example, apolynucleotide of the disclosure can be engineered to include miR-192and miR-122 to regulate expression of the polynucleotide in the liverand kidneys of a subject. In another embodiment, a polynucleotide of thedisclosure can be engineered to include more than one miRNA site for thesame tissue.

In some embodiments, the therapeutic window and or differentialexpression associated with the polypeptide encoded by a polynucleotideof the disclosure can be altered with a miRNA binding site. For example,a polynucleotide encoding a polypeptide that provides a death signal canbe designed to be more highly expressed in cancer cells by virtue of themiRNA signature of those cells. Where a cancer cell expresses a lowerlevel of a particular miRNA, the polynucleotide encoding the bindingsite for that miRNA (or miRNAs) would be more highly expressed. Hence,the polypeptide that provides a death signal triggers or induces celldeath in the cancer cell. Neighboring noncancer cells, harboring ahigher expression of the same miRNA would be less affected by theencoded death signal as the polynucleotide would be expressed at a lowerlevel due to the effects of the miRNA binding to the binding site or“sensor” encoded in the 3′UTR. Conversely, cell survival orcytoprotective signals can be delivered to tissues containing cancer andnon-cancerous cells where a miRNA has a higher expression in the cancercells—the result being a lower survival signal to the cancer cell and alarger survival signal to the normal cell. Multiple polynucleotides canbe designed and administered having different signals based on the useof miRNA binding sites as described herein.

In some embodiments, the expression of a polynucleotide of thedisclosure can be controlled by incorporating at least one sensorsequence in the polynucleotide and formulating the polynucleotide foradministration. As a non-limiting example, a polynucleotide of thedisclosure can be targeted to a tissue or cell by incorporating a miRNAbinding site and formulating the polynucleotide in a lipid nanoparticlecomprising a cationic lipid, including any of the lipids describedherein.

A polynucleotide of the disclosure can be engineered for more targetedexpression in specific tissues, cell types, or biological conditionsbased on the expression patterns of miRNAs in the different tissues,cell types, or biological conditions. Through introduction oftissue-specific miRNA binding sites, a polynucleotide of the disclosurecan be designed for optimal protein expression in a tissue or cell, orin the context of a biological condition.

In some embodiments, a polynucleotide of the disclosure can be designedto incorporate miRNA binding sites that either have 100% identity toknown miRNA seed sequences or have less than 100% identity to miRNA seedsequences. In some embodiments, a polynucleotide of the disclosure canbe designed to incorporate miRNA binding sites that have at least: 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity toknown miRNA seed sequences. The miRNA seed sequence can be partiallymutated to decrease miRNA binding affinity and as such result in reduceddownmodulation of the polynucleotide. In essence, the degree of match ormis-match between the miRNA binding site and the miRNA seed can act as arheostat to more finely tune the ability of the miRNA to modulateprotein expression. In addition, mutation in the non-seed region of amiRNA binding site can also impact the ability of a miRNA to modulateprotein expression.

In one embodiment, a miRNA sequence can be incorporated into the loop ofa stem loop.

In another embodiment, a miRNA seed sequence can be incorporated in theloop of a stem loop and a miRNA binding site can be incorporated intothe 5′ or 3′ stem of the stem loop.

In one embodiment, a translation enhancer element (TEE) can beincorporated on the 5′end of the stem of a stem loop and a miRNA seedcan be incorporated into the stem of the stem loop. In anotherembodiment, a TEE can be incorporated on the 5′ end of the stem of astem loop, a miRNA seed can be incorporated into the stem of the stemloop and a miRNA binding site can be incorporated into the 3′ end of thestem or the sequence after the stem loop. The miRNA seed and the miRNAbinding site can be for the same and/or different miRNA sequences.

In one embodiment, the incorporation of a miRNA sequence and/or a TEEsequence changes the shape of the stem loop region which can increaseand/or decrease translation. (see e.g, Kedde et al., “A Pumilio-inducedRNA structure switch in p27-3′UTR controls miR-221 and miR-22accessibility.” Nature Cell Biology. 2010, incorporated herein byreference in its entirety).

In one embodiment, the 5′-UTR of a polynucleotide of the disclosure cancomprise at least one miRNA sequence. The miRNA sequence can be, but isnot limited to, a 19 or 22 nucleotide sequence and/or a miRNA sequencewithout the seed.

In one embodiment the miRNA sequence in the 5′UTR can be used tostabilize a polynucleotide of the disclosure described herein.

In another embodiment, a miRNA sequence in the 5′UTR of a polynucleotideof the disclosure can be used to decrease the accessibility of the siteof translation initiation such as, but not limited to a start codon.See, e.g., Matsuda et al., PLoS One. 2010 11(5):e15057; incorporatedherein by reference in its entirety, which used antisense locked nucleicacid (LNA) oligonucleotides and exon-junction complexes (EJCs) around astart codon (−4 to +37 where the A of the AUG codons is +1) in order todecrease the accessibility to the first start codon (AUG). Matsudashowed that altering the sequence around the start codon with an LNA orEJC affected the efficiency, length and structural stability of apolynucleotide. A polynucleotide of the disclosure can comprise a miRNAsequence, instead of the LNA or EJC sequence described by Matsuda et al,near the site of translation initiation in order to decrease theaccessibility to the site of translation initiation. The site oftranslation initiation can be prior to, after or within the miRNAsequence. As a non-limiting example, the site of translation initiationcan be located within a miRNA sequence such as a seed sequence orbinding site. As another non-limiting example, the site of translationinitiation can be located within a miR-122 sequence such as the seedsequence or the mir-122 binding site.

In some embodiments, a polynucleotide of the disclosure can include atleast one miRNA in order to dampen the antigen presentation by antigenpresenting cells. The miRNA can be the complete miRNA sequence, themiRNA seed sequence, the miRNA sequence without the seed, or acombination thereof. As a non-limiting example, a miRNA incorporatedinto a polynucleotide of the disclosure can be specific to thehematopoietic system. As another non-limiting example, a miRNAincorporated into a polynucleotide of the disclosure to dampen antigenpresentation is miR-142-3p.

In some embodiments, a polynucleotide of the disclosure can include atleast one miRNA in order to dampen expression of the encoded polypeptidein a tissue or cell of interest. As a non-limiting example, apolynucleotide of the disclosure can include at least one miR-122binding site in order to dampen expression of an encoded polypeptide ofinterest in the liver. As another non-limiting example a polynucleotideof the disclosure can include at least one miR-142-3p binding site,miR-142-3p seed sequence, miR-142-3p binding site without the seed,miR-142-5p binding site, miR-142-5p seed sequence, miR-142-5p bindingsite without the seed, miR-146 binding site, miR-146 seed sequenceand/or miR-146 binding site without the seed sequence.

In some embodiments, a polynucleotide of the disclosure can comprise atleast one miRNA binding site in the 3′UTR in order to selectivelydegrade mRNA therapeutics in the immune cells to subdue unwantedimmunogenic reactions caused by therapeutic delivery. As a non-limitingexample, the miRNA binding site can make a polynucleotide of thedisclosure more unstable in antigen presenting cells. Non-limitingexamples of these miRNAs include mir-142-5p, mir-142-3p, mir-146a-5p,and mir-146-3p.

In one embodiment, a polynucleotide of the disclosure comprises at leastone miRNA sequence in a region of the polynucleotide that can interactwith a RNA binding protein.

In some embodiments, the polynucleotide of the disclosure (e.g., a RNA,e.g., a mRNA) comprising (i) a sequence-optimized nucleotide sequence(e.g., an ORF) and (ii) a miRNA binding site (e.g., a miRNA binding sitethat binds to miR-142).

In some embodiments, the polynucleotide of the disclosure comprises auracil-modified sequence encoding a polypeptide disclosed herein and amiRNA binding site disclosed herein, e.g., a miRNA binding site thatbinds to miR-142 or miR-122. In some embodiments, the uracil-modifiedsequence encoding a polypeptide comprises at least one chemicallymodified nucleobase, e.g., 5-methoxyuracil. In some embodiments, atleast 95% of a type of nucleobase (e.g., uracil) in a uracil-modifiedsequence encoding a polypeptide of the disclosure are modifiednucleobases. In some embodiments, at least 95% of uricil in auracil-modified sequence encoding a polypeptide is 5-methoxyuridine. Insome embodiments, the polynucleotide comprising a nucleotide sequenceencoding a polypeptide disclosed herein and a miRNA binding site isformulated with a delivery agent, e.g., a compound having the Formula(I), e.g., any of Compounds 1-147.

Modified Polynucleotides Comprising Functional RNA Elements

The present disclosure provides synthetic polynucleotides comprising amodification (e.g., an RNA element), wherein the modification provides adesired translational regulatory activity. In some embodiments, thedisclosure provides a polynucleotide comprising a 5′ untranslated region(UTR), an initiation codon, a full open reading frame encoding apolypeptide, a 3′ UTR, and at least one modification, wherein the atleast one modification provides a desired translational regulatoryactivity, for example, a modification that promotes and/or enhances thetranslational fidelity of mRNA translation. In some embodiments, thedesired translational regulatory activity is a cis-acting regulatoryactivity. In some embodiments, the desired translational regulatoryactivity is an increase in the residence time of the 43S pre-initiationcomplex (PIC) or ribosome at, or proximal to, the initiation codon. Insome embodiments, the desired translational regulatory activity is anincrease in the initiation of polypeptide synthesis at or from theinitiation codon. In some embodiments, the desired translationalregulatory activity is an increase in the amount of polypeptidetranslated from the full open reading frame. In some embodiments, thedesired translational regulatory activity is an increase in the fidelityof initiation codon decoding by the PIC or ribosome. In someembodiments, the desired translational regulatory activity is inhibitionor reduction of leaky scanning by the PIC or ribosome. In someembodiments, the desired translational regulatory activity is a decreasein the rate of decoding the initiation codon by the PIC or ribosome. Insome embodiments, the desired translational regulatory activity isinhibition or reduction in the initiation of polypeptide synthesis atany codon within the mRNA other than the initiation codon. In someembodiments, the desired translational regulatory activity is inhibitionor reduction of the amount of polypeptide translated from any openreading frame within the mRNA other than the full open reading frame. Insome embodiments, the desired translational regulatory activity isinhibition or reduction in the production of aberrant translationproducts. In some embodiments, the desired translational regulatoryactivity is a combination of one or more of the foregoing translationalregulatory activities.

Accordingly, the present disclosure provides a polynucleotide, e.g., anmRNA, comprising an RNA element that comprises a sequence and/or an RNAsecondary structure(s) that provides a desired translational regulatoryactivity as described herein. In some aspects, the mRNA comprises an RNAelement that comprises a sequence and/or an RNA secondary structure(s)that promotes and/or enhances the translational fidelity of mRNAtranslation. In some aspects, the mRNA comprises an RNA element thatcomprises a sequence and/or an RNA secondary structure(s) that providesa desired translational regulatory activity, such as inhibiting and/orreducing leaky scanning. In some aspects, the disclosure provides anmRNA that comprises an RNA element that comprises a sequence and/or anRNA secondary structure(s) that inhibits and/or reduces leaky scanningthereby promoting the translational fidelity of the mRNA.

In some embodiments, the RNA element comprises natural and/or modifiednucleotides. In some embodiments, the RNA element comprises of asequence of linked nucleotides, or derivatives or analogs thereof, thatprovides a desired translational regulatory activity as describedherein. In some embodiments, the RNA element comprises a sequence oflinked nucleotides, or derivatives or analogs thereof, that forms orfolds into a stable RNA secondary structure, wherein the RNA secondarystructure provides a desired translational regulatory activity asdescribed herein. RNA elements can be identified and/or characterizedbased on the primary sequence of the element (e.g., GC-rich element), byRNA secondary structure formed by the element (e.g. stem-loop), by thelocation of the element within the RNA molecule (e.g., located withinthe 5′ UTR of an mRNA), by the biological function and/or activity ofthe element (e.g., “translational enhancer element”), and anycombination thereof.

In some aspects, the disclosure provides an mRNA having one or morestructural modifications that inhibits leaky scanning and/or promotesthe translational fidelity of mRNA translation, wherein at least one ofthe structural modifications is a GC-rich RNA element. In some aspects,the disclosure provides a modified mRNA comprising at least onemodification, wherein at least one modification is a GC-rich RNA elementcomprising a sequence of linked nucleotides, or derivatives or analogsthereof, preceding a Kozak consensus sequence in a 5′ UTR of the mRNA.In one embodiment, the GC-rich RNA element is located about 30, about25, about 20, about 15, about 10, about 5, about 4, about 3, about 2, orabout 1 nucleotide(s) upstream of a Kozak consensus sequence in the 5′UTR of the mRNA. In another embodiment, the GC-rich RNA element islocated 15-30, 15-20, 15-25, 10-15, or 5-10 nucleotides upstream of aKozak consensus sequence. In another embodiment, the GC-rich RNA elementis located immediately adjacent to a Kozak consensus sequence in the 5′UTR of the mRNA.

In any of the foregoing or related aspects, the disclosure provides aGC-rich RNA element which comprises a sequence of 3-30, 5-25, 10-20,15-20, about 20, about 15, about 12, about 10, about 7, about 6 or about3 nucleotides, derivatives or analogs thereof, linked in any order,wherein the sequence composition is 70-80% cytosine, 60-70% cytosine,50%-60% cytosine, 40-50% cytosine, 30-40% cytosine bases. In any of theforegoing or related aspects, the disclosure provides a GC-rich RNAelement which comprises a sequence of 3-30, 5-25, 10-20, 15-20, about20, about 15, about 12, about 10, about 7, about 6 or about 3nucleotides, derivatives or analogs thereof, linked in any order,wherein the sequence composition is about 80% cytosine, about 70%cytosine, about 60% cytosine, about 50% cytosine, about 40% cytosine, orabout 30% cytosine.

In any of the foregoing or related aspects, the disclosure provides aGC-rich RNA element which comprises a sequence of 20, 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 nucleotides, orderivatives or analogs thereof, linked in any order, wherein thesequence composition is 70-80% cytosine, 60-70% cytosine, 50%-60%cytosine, 40-50% cytosine, or 30-40% cytosine. In any of the foregoingor related aspects, the disclosure provides a GC-rich RNA element whichcomprises a sequence of 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9,8, 7, 6, 5, 4, or 3 nucleotides, or derivatives or analogs thereof,linked in any order, wherein the sequence composition is about 80%cytosine, about 70% cytosine, about 60% cytosine, about 50% cytosine,about 40% cytosine, or about 30% cytosine.

In some embodiments, the disclosure provides a modified mRNA comprisingat least one modification, wherein at least one modification is aGC-rich RNA element comprising a sequence of linked nucleotides, orderivatives or analogs thereof, preceding a Kozak consensus sequence ina 5′ UTR of the mRNA, wherein the GC-rich RNA element is located about30, about 25, about 20, about 15, about 10, about 5, about 4, about 3,about 2, or about 1 nucleotide(s) upstream of a Kozak consensus sequencein the 5′ UTR of the mRNA, and wherein the GC-rich RNA element comprisesa sequence of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20 nucleotides, or derivatives or analogs thereof, linked in anyorder, wherein the sequence composition is >50% cytosine. In someembodiments, the sequence composition is >55% cytosine, >60%cytosine, >65% cytosine, >70% cytosine, >75% cytosine, >80%cytosine, >85% cytosine, or >90% cytosine.

In other aspects, the disclosure provides a modified mRNA comprising atleast one modification, wherein at least one modification is a GC-richRNA element comprising a sequence of linked nucleotides, or derivativesor analogs thereof, preceding a Kozak consensus sequence in a 5′ UTR ofthe mRNA, wherein the GC-rich RNA element is located about 30, about 25,about 20, about 15, about 10, about 5, about 4, about 3, about 2, orabout 1 nucleotide(s) upstream of a Kozak consensus sequence in the 5′UTR of the mRNA, and wherein the GC-rich RNA element comprises asequence of about 3-30, 5-25, 10-20, 15-20 or about 20, about 15, about12, about 10, about 6 or about 3 nucleotides, or derivatives oranalogues thereof, wherein the sequence comprises a repeating GC-motif,wherein the repeating GC-motif is [CCG]n, wherein n=1 to 10, n=2 to 8,n=3 to 6, or n=4 to 5. In some embodiments, the sequence comprises arepeating GC-motif [CCG]n, wherein n=1, 2, 3, 4 or 5. In someembodiments, the sequence comprises a repeating GC-motif [CCG]n, whereinn=1, 2, or 3. In some embodiments, the sequence comprises a repeatingGC-motif [CCG]n, wherein n=1. In some embodiments, the sequencecomprises a repeating GC-motif [CCG]n, wherein n=2. In some embodiments,the sequence comprises a repeating GC-motif [CCG]n, wherein n=3. In someembodiments, the sequence comprises a repeating GC-motif [CCG]n, whereinn=4 (SEQ ID NO: 177). In some embodiments, the sequence comprises arepeating GC-motif [CCG]n, wherein n=5 (SEQ ID NO: 178).

In another aspect, the disclosure provides a modified mRNA comprising atleast one modification, wherein at least one modification is a GC-richRNA element comprising a sequence of linked nucleotides, or derivativesor analogs thereof, preceding a Kozak consensus sequence in a 5′ UTR ofthe mRNA, wherein the GC-rich RNA element comprises any one of thesequences set forth in Table 4. In one embodiment, the GC-rich RNAelement is located about 30, about 25, about 20, about 15, about 10,about 5, about 4, about 3, about 2, or about 1 nucleotide(s) upstream ofa Kozak consensus sequence in the 5′ UTR of the mRNA. In anotherembodiment, the GC-rich RNA element is located about 15-30, 15-20,15-25, 10-15, or 5-10 nucleotides upstream of a Kozak consensussequence. In another embodiment, the GC-rich RNA element is locatedimmediately adjacent to a Kozak consensus sequence in the 5′ UTR of themRNA.

In other aspects, the disclosure provides a modified mRNA comprising atleast one modification, wherein at least one modification is a GC-richRNA element comprising the sequence V1 [CCCCGGCGCC] (SEQ ID NO: 179) asset forth in Table 4, or derivatives or analogs thereof, preceding aKozak consensus sequence in the 5′ UTR of the mRNA. In some embodiments,the GC-rich element comprises the sequence V1 as set forth in Table 4located immediately adjacent to and upstream of the Kozak consensussequence in the 5′ UTR of the mRNA. In some embodiments, the GC-richelement comprises the sequence V1 as set forth in Table 4 located 1, 2,3, 4, 5, 6, 7, 8, 9 or 10 bases upstream of the Kozak consensus sequencein the 5′ UTR of the mRNA. In other embodiments, the GC-rich elementcomprises the sequence V1 as set forth in Table 4 located 1-3, 3-5, 5-7,7-9, 9-12, or 12-15 bases upstream of the Kozak consensus sequence inthe 5′ UTR of the mRNA.

In other aspects, the disclosure provides a modified mRNA comprising atleast one modification, wherein at least one modification is a GC-richRNA element comprising the sequence V2 [CCCCGGC] as set forth in Table4, or derivatives or analogs thereof, preceding a Kozak consensussequence in the 5′ UTR of the mRNA. In some embodiments, the GC-richelement comprises the sequence V2 as set forth in Table 4 locatedimmediately adjacent to and upstream of the Kozak consensus sequence inthe 5′ UTR of the mRNA. In some embodiments, the GC-rich elementcomprises the sequence V2 as set forth in Table 4 located 1, 2, 3, 4, 5,6, 7, 8, 9 or 10 bases upstream of the Kozak consensus sequence in the5′ UTR of the mRNA. In other embodiments, the GC-rich element comprisesthe sequence V2 as set forth in Table 4 located 1-3, 3-5, 5-7, 7-9,9-12, or 12-15 bases upstream of the Kozak consensus sequence in the 5′UTR of the mRNA.

In other aspects, the disclosure provides a modified mRNA comprising atleast one modification, wherein at least one modification is a GC-richRNA element comprising the sequence EK [GCCGCC] as set forth in Table 4,or derivatives or analogs thereof, preceding a Kozak consensus sequencein the 5′ UTR of the mRNA. In some embodiments, the GC-rich elementcomprises the sequence EK as set forth in Table 4 located immediatelyadjacent to and upstream of the Kozak consensus sequence in the 5′ UTRof the mRNA. In some embodiments, the GC-rich element comprises thesequence EK as set forth in Table 4 located 1, 2, 3, 4, 5, 6, 7, 8, 9 or10 bases upstream of the Kozak consensus sequence in the 5′ UTR of themRNA. In other embodiments, the GC-rich element comprises the sequenceEK as set forth in Table 4 located 1-3, 3-5, 5-7, 7-9, 9-12, or 12-15bases upstream of the Kozak consensus sequence in the 5′ UTR of themRNA.

In yet other aspects, the disclosure provides a modified mRNA comprisingat least one modification, wherein at least one modification is aGC-rich RNA element comprising the sequence V1[CCCCGGCGCC] (SEQ ID NO:179) as set forth in Table 4, or derivatives or analogs thereof,preceding a Kozak consensus sequence in the 5′ UTR of the mRNA, whereinthe 5′ UTR comprises the following sequence shown in Table 4:

(SEQ ID NO: 180) GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGA.

In some embodiments, the GC-rich element comprises the sequence V1 asset forth in Table 4 located immediately adjacent to and upstream of theKozak consensus sequence in the 5′ UTR sequence shown in Table 4. Insome embodiments, the GC-rich element comprises the sequence V1 as setforth in Table 4 located 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 bases upstreamof the Kozak consensus sequence in the 5′ UTR of the mRNA, wherein the5′ UTR comprises the following sequence shown in Table 4:

(SEQ ID NO: 180) GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGA.

In other embodiments, the GC-rich element comprises the sequence V1 asset forth in Table 4 located 1-3, 3-5, 5-7, 7-9, 9-12, or 12-15 basesupstream of the Kozak consensus sequence in the 5′ UTR of the mRNA,wherein the 5′ UTR comprises the following sequence shown in Table 4:

(SEQ ID NO: 180) GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGA.

In some embodiments, the 5′ UTR comprises the following sequence setforth in Table 4:

(SEQ ID NO: 181) GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGACCCCGGCGCCGCCACCIn some embodiments, the 5′ UTR comprises the following sequence setforth in Table 4:

(SEQ ID NO: 182) GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGACCCCGGCGC CACC

TABLE 4 SEQ ID NO: 5′ UTRs 5′UTR Sequence 176 StandardGGGAAATAAGAGAGAAAAGAAGAGTAAGAA GAAATATAAGAGCCACC 180 UTRGGGAAATAAGAGAGAAAAGAAGAGTAAGA AGAAATATAAGA 181 V1-UTRGGGAAATAAGAGAGAAAAGAAGAGTAAGAA GAAATATAAGACCCCGGCGCCGCCACC 182 V2-UTRGGGAAATAAGAGAGAAAAGAAGAGTAAGAA GAAATATAAGACCCCGGCGCCACC SEQ ID NO:GC-Rich RNA Elements Sequence K0 (Traditional Kozak [GCCA/GCC]consensus) EK [GCCGCC] 179 V1 [CCCCGGCGCC] V2 [CCCCGGC] (CCG)_(n), wheren = 1-10 [CCG]_(n) (GCC)_(n), where n = 1-10 [GCC]_(n) 177 (CCG)_(n),where n = 4 [CCGCCGCCGCCG] 178 (CCG)_(n), where n = 5 [CCGCCGCCGCCGCCG]

In another aspect, the disclosure provides a modified mRNA comprising atleast one modification, wherein at least one modification is a GC-richRNA element comprising a stable RNA secondary structure comprising asequence of nucleotides, or derivatives or analogs thereof, linked in anorder which forms a hairpin or a stem-loop. In one embodiment, thestable RNA secondary structure is upstream of the Kozak consensussequence. In another embodiment, the stable RNA secondary structure islocated about 30, about 25, about 20, about 15, about 10, or about 5nucleotides upstream of the Kozak consensus sequence. In anotherembodiment, the stable RNA secondary structure is located about 20,about 15, about 10 or about 5 nucleotides upstream of the Kozakconsensus sequence. In another embodiment, the stable RNA secondarystructure is located about 5, about 4, about 3, about 2, about 1nucleotides upstream of the Kozak consensus sequence. In anotherembodiment, the stable RNA secondary structure is located about 15-30,about 15-20, about 15-25, about 10-15, or about 5-10 nucleotidesupstream of the Kozak consensus sequence. In another embodiment, thestable RNA secondary structure is located 12-15 nucleotides upstream ofthe Kozak consensus sequence. In another embodiment, the stable RNAsecondary structure has a deltaG of about −30 kcal/mol, about −20 to −30kcal/mol, about −20 kcal/mol, about −10 to −20 kcal/mol, about −10kcal/mol, about −5 to −10 kcal/mol.

In another embodiment, the modification is operably linked to an openreading frame encoding a polypeptide and wherein the modification andthe open reading frame are heterologous.

In another embodiment, the sequence of the GC-rich RNA element iscomprised exclusively of guanine (G) and cytosine (C) nucleobases.

RNA elements that provide a desired translational regulatory activity asdescribed herein can be identified and characterized using knowntechniques, such as ribosome profiling. Ribosome profiling is atechnique that allows the determination of the positions of PICs and/orribosomes bound to mRNAs (see e.g., Ingolia et al., (2009) Science324(5924):218-23, incorporated herein by reference). The technique isbased on protecting a region or segment of mRNA, by the PIC and/orribosome, from nuclease digestion. Protection results in the generationof a 30-bp fragment of RNA termed a ‘footprint’. The sequence andfrequency of RNA footprints can be analyzed by methods known in the art(e.g., RNA-seq). The footprint is roughly centered on the A-site of theribosome. If the PIC or ribosome dwells at a particular position orlocation along an mRNA, footprints generated at these position would berelatively common. Studies have shown that more footprints are generatedat positions where the PIC and/or ribosome exhibits decreasedprocessivity and fewer footprints where the PIC and/or ribosome exhibitsincreased processivity (Gardin et al., (2014) eLife 3:e03735). In someembodiments, residence time or the time of occupancy of a the PIC orribosome at a discrete position or location along an polynucleotidecomprising any one or more of the RNA elements described herein isdetermined by ribosome profiling.

Preparation of High Purity RNA

In order to enhance the purity of synthetically produced RNA, modifiedin vitro transcription (IVT) processes which produce RNA preparationshaving vastly different properties from RNA produced using a traditionalIVT process may be used. The RNA preparations produced according tothese methods have properties that enable the production ofqualitatively and quantitatively superior compositions. Even whencoupled with extensive purification processes, RNA produced usingtraditional IVT methods is qualitatively and quantitatively distinctfrom the RNA preparations produced by the modified IVT processes. Forinstance, the purified RNA preparations are less immunogenic incomparison to RNA preparations made using traditional IVT. Additionally,increased protein expression levels with higher purity are produced fromthe purified RNA preparations.

Traditional IVT reactions are performed by incubating a DNA templatewith an RNA polymerase and equimolar quantities of nucleotidetriphosphates, including GTP, ATP, CTP, and UTP in a transcriptionbuffer. An RNA transcript having a 5′ terminal guanosine triphosphate isproduced from this reaction. These reactions also result in theproduction of a number of impurities such as double stranded and singlestranded RNAs which are immunostimulatory and may have an additiveimpact. The purity methods described herein prevent formation of reversecomplements and thus prevent the innate immune recognition of bothspecies. In some embodiments the modified IVT methods result in theproduction of RNA having significantly reduced T cell activity than anRNA preparation made using prior art methods with equimolar NTPs. Theprior art attempts to remove these undesirable components using a seriesof subsequent purification steps. Such purification methods areundesirable because they involve additional time and resources and alsoresult in the incorporation of residual organic solvents in the finalproduct, which is undesirable for a pharmaceutical product. It is laborand capital intensive to scale up processes like reverse phasechromatography (RP): utilizing for instance explosion proof facilities,HPLC columns and purification systems rated for high pressure, hightemperature, flammable solvents etc. The scale and throughput for largescale manufacture are limited by these factors. Subsequent purificationis also required to remove alkylammonium ion pair utilized in RPprocess. In contrast the methods described herein even enhance currentlyutilized methods (eg RP). Lower impurity load leads to higherpurification recovery of full length RNA devoid of cytokine inducingcontaminants eg. higher quality of materials at the outset.

The modified IVT methods involve the manipulation of one or more of thereaction parameters in the IVT reaction to produce a RNA preparation ofhighly functional RNA without one or more of the undesirablecontaminants produced using the prior art processes. One parameter inthe IVT reaction that may be manipulated is the relative amount of anucleotide or nucleotide analog in comparison to one or more othernucleotides or nucleotide analogs in the reaction mixture (e.g.,disparate nucleotide amounts or concentration). For instance, the IVTreaction may include an excess of a nucleotides, e.g., nucleotidemonophosphate, nucleotide diphosphate or nucleotide triphosphate and/oran excess of nucleotide analogs and/or nucleoside analogs. The methodsproduce a high yield product which is significantly more pure thanproducts produced by traditional IVT methods.

Nucleotide analogs are compounds that have the general structure of anucleotide or are structurally similar to a nucleotide or portionthereof. In particular, nucleotide analogs are nucleotides whichcontain, for example, an analogue of the nucleic acid portion, sugarportion and/or phosphate groups of the nucleotide. Nucleotides include,for instance, nucleotide monophosphates, nucleotide diphosphates, andnucleotide triphosphates. A nucleotide analog, as used herein isstructurally similar to a nucleotide or portion thereof but does nothave the typical nucleotide structure (nucleobase-ribose-phosphate).Nucleoside analogs are compounds that have the general structure of anucleoside or are structurally similar to a nucleoside or portionthereof. In particular, nucleoside analogs are nucleosides whichcontain, for example, an analogue of the nucleic acid and/or sugarportion of the nucleoside.

The nucleotide analogs useful in the methods are structurally similar tonucleotides or portions thereof but, for example, are not polymerizableby T7. Nucleotide/nucleoside analogs as used herein (including C, T, A,U, G, dC, dT, dA, dU, or dG analogs) include for instance, antiviralnucleotide analogs, phosphate analogs (soluble or immobilized,hydrolyzable or non-hydrolyzable), dinucleotide, trinucleotide,tetranucleotide, e.g., a cap analog, or a precursor/substrate forenzymatic capping (vaccinia, or ligase), a nucleotide labelled with afunctional group to facilitate ligation/conjugation of cap or 5′ moiety(IRES), a nucleotide labelled with a 5′ PO4 to facilitate ligation ofcap or 5′ moiety, or a nucleotide labelled with a functionalgroup/protecting group that can be chemically or enzymaticallycleavable. Antiviral nucleotide/nucleoside analogs include but are notlimited to Ganciclovir, Entecavir, Telbivudine, Vidarabine andCidofovir.

The IVT reaction typically includes the following: an RNA polymerase,e.g., a T7 RNA polymerase at a final concentration of, e.g., 1000-12000U/mL, e.g., 7000 U/mL; the DNA template at a final concentration of,e.g., 10-70 nM, e.g., 40 nM; nucleotides (NTPs) at a final concentrationof e.g., 0.5-10 mM, e.g., 7.5 mM each; magnesium at a finalconcentration of, e.g., 12-60 mM, e.g., magnesium acetate at 40 mM; abuffer such as, e.g., HEPES or Tris at a pH of, e.g., 7-8.5, e.g. 40 mMTris HCl, pH 8. In some embodiments 5 mM dithiothreitol (DTT) and/or 1mM spermidine may be included. In some embodiments, an RNase inhibitoris included in the IVT reaction to ensure no RNase induced degradationduring the transcription reaction. For example, murine RNase inhibitorcan be utilized at a final concentration of 1000 U/mL. In someembodiments a pyrophosphatase is included in the IVT reaction to cleavethe inorganic pyrophosphate generated following each nucleotideincorporation into two units of inorganic phosphate. This ensures thatmagnesium remains in solution and does not precipitate as magnesiumpyrophosphate. For example, an E. coli inorganic pyrophosphatase can beutilized at a final concentration of 1 U/mL.

Similar to traditional methods, the modified method may also be producedby forming a reaction mixture comprising a DNA template, and one or moreNTPs such as ATP, CTP, UTP, GTP (or corresponding analog ofaforementioned components) and a buffer. The reaction is then incubatedunder conditions such that the RNA is transcribed. However, the modifiedmethods utilize the presence of an excess amount of one or morenucleotides and/or nucleotide analogs that can have significant impacton the end product. These methods involve a modification in the amount(e.g., molar amount or quantity) of nucleotides and/or nucleotideanalogs in the reaction mixture. In some aspects, one or morenucleotides and/or one or more nucleotide analogs may be added in excessto the reaction mixture. An excess of nucleotides and/or nucleotideanalogs is any amount greater than the amount of one or more of theother nucleotides such as NTPs in the reaction mixture. For instance, anexcess of a nucleotide and/or nucleotide analog may be a greater amountthan the amount of each or at least one of the other individual NTPs inthe reaction mixture or may refer to an amount greater than equimolaramounts of the other NTPs.

In the embodiment when the nucleotide and/or nucleotide analog that isincluded in the reaction mixture is an NTP, the NTP may be present in ahigher concentration than all three of the other NTPs included in thereaction mixture. The other three NTPs may be in an equimolarconcentration to one another. Alternatively one or more of the threeother NTPs may be in a different concentration than one or more of theother NTPs.

Thus, in some embodiments the IVT reaction may include an equimolaramount of nucleotide triphosphate relative to at least one of the othernucleotide triphosphates.

In some embodiments the RNA is produced by a process or is preparable bya process comprising

(a) forming a reaction mixture comprising a DNA template and NTPsincluding adenosine triphosphate (ATP), cytidine triphosphate (CTP),uridine triphosphate (UTP), guanosine triphosphate (GTP) and optionallyguanosine diphosphate (GDP), and (eg. buffer containing T7 co-factor eg.magnesium).

(b) incubating the reaction mixture under conditions such that the RNAis transcribed,

wherein the concentration of at least one of GTP, CTP, ATP, and UTP isat least 2× greater than the concentration of any one or more of ATP,CTP or UTP or the reaction further comprises a nucleotide analog andwherein the concentration of the nucleotide analog is at least 2×greater than the concentration of any one or more of ATP, CTP or UTP.

In some embodiments the ratio of concentration of GTP to theconcentration of any one ATP, CTP or UTP is at least 2:1, at least 3:1,at least 4:1, at least 5:1 or at least 6:1. The ratio of concentrationof GTP to concentration of ATP, CTP and UTP is, in some embodiments 2:1,4:1 and 4:1, respectively. In other embodiments the ratio ofconcentration of GTP to concentration of ATP, CTP and UTP is 3:1, 6:1and 6:1, respectively. The reaction mixture may comprise GTP and GDP andwherein the ratio of concentration of GTP plus GDP to the concentrationof any one of ATP, CTP or UTP is at least 2:1, at least 3:1, at least4:1, at least 5:1 or at least 6:1 In some embodiments the ratio ofconcentration of GTP plus GDP to concentration of ATP, CTP and UTP is3:1, 6:1 and 6:1, respectively.

In some embodiments the method involves incubating the reaction mixtureunder conditions such that the RNA is transcribed, wherein the effectiveconcentration of phosphate in the reaction is at least 150 mM phosphate,at least 160 mM, at least 170 mM, at least 180 mM, at least 190 mM, atleast 200 mM, at least 210 mM or at least 220 mM. The effectiveconcentration of phosphate in the reaction may be 180 mM. The effectiveconcentration of phosphate in the reaction in some embodiments is 195mM. In other embodiments the effective concentration of phosphate in thereaction is 225 mM.

In other embodiments the RNA is produced by a process or is preparableby a process comprising wherein a buffer magnesium-containing buffer isused when forming the reaction mixture comprising a DNA template andATP, CTP, UTP, GTP. In some embodiments the magnesium-containing buffercomprises Mg2+ and wherein the molar ratio of concentration of ATP plusCTP plus UTP pus GTP to concentration of Mg2+ is at least 1.0, at least1.25, at least 1.5, at least 1.75, at least 1.85, at least 3 or higher.The molar ratio of concentration of ATP plus CTP plus UTP pus GTP toconcentration of Mg2+ may be 1.5. The molar ratio of concentration ofATP plus CTP plus UTP pus GTP to concentration of Mg2+ in someembodiments is 1.88. The molar ratio of concentration of ATP plus CTPplus UTP pus GTP to concentration of Mg2+ in some embodiments is 3.

In some embodiments the composition is produced by a process which doesnot comprise an dsRNase (e.g., RNaseII) treatment step. In otherembodiments the composition is produced by a process which does notcomprise a reverse phase (RP) chromatography purification step. In yetother embodiments the composition is produced by a process which doesnot comprise a high-performance liquid chromatography (HPLC)purification step.

In some embodiments the ratio of concentration of GTP to theconcentration of any one ATP, CTP or UTP is at least 2:1, at least 3:1,at least 4:1, at least 5:1 or at least 6:1 to produce the RNA.

The purity of the products may be assessed using known analyticalmethods and assays. For instance, the amount of reverse complementtranscription product or cytokine-inducing RNA contaminant may bedetermined by high-performance liquid chromatography (such asreverse-phase chromatography, size-exclusion chromatography),Bioanalyzer chip-based electrophoresis system, ELISA, flow cytometry,acrylamide gel, a reconstitution or surrogate type assay. The assays maybe performed with or without nuclease treatment (P1, RNase III, RNase Hetc.) of the RNA preparation. Electrophoretic/chromatographic/mass specanalysis of nuclease digestion products may also be performed.

In some embodiments the purified RNA preparations comprise contaminanttranscripts that have a length less than a full length transcript, suchas for instance at least 100, 200, 300, 400, 500, 600, 700, 800, or 900nucleotides less than the full length. Contaminant transcripts caninclude reverse or forward transcription products (transcripts) thathave a length less than a full length transcript, such as for instanceat least 100, 200, 300, 400, 500, 600, 700, 800, or 900 nucleotides lessthan the full length. Exemplary forward transcripts include, forinstance, abortive transcripts. In certain embodiments the compositioncomprises a tri-phosphate poly-U reverse complement of less than 30nucleotides. In some embodiments the composition comprises atri-phosphate poly-U reverse complement of any length hybridized to afull length transcript. In other embodiments the composition comprises asingle stranded tri-phosphate forward transcript. In other embodimentsthe composition comprises a single stranded RNA having a terminaltri-phosphate-G. In other embodiments the composition comprises singleor double stranded RNA of less than 12 nucleotides or base pairs(including forward or reverse complement transcripts). In any of theseembodiments the composition may include less than 50%, 45%, 40%, 35%,30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.5% ofany one of or combination of these less than full length transcripts.

Delivery Vehicles General

The mRNAs of the disclosure may be formulated in nanoparticles or otherdelivery vehicles, e.g., to protect them from degradation when deliveredto a subject. Illustrative nanoparticles are described in Panyam, J. &Labhasetwar, V. Adv. Drug Deliv. Rev. 55, 329-347 (2003) and Peer, D. etal. Nature Nanotech. 2, 751-760 (2007). In certain embodiments, an mRNAof the disclosure is encapsulated within a nanoparticle. In particularembodiments, a nanoparticle is a particle having at least one dimension(e.g., a diameter) less than or equal to 1000 nM, less than or equal to500 nM or less than or equal to 100 nM. In particular embodiments, ananoparticle includes a lipid. Lipid nanoparticles include, but are notlimited to, liposomes and micelles. Any of a number of lipids may bepresent, including cationic and/or ionizable lipids, anionic lipids,neutral lipids, amphipathic lipids, PEGylated lipids, and/or structurallipids. Such lipids can be used alone or in combination. In particularembodiments, a lipid nanoparticle comprises one or more mRNAs describedherein.

In some embodiments, the lipid nanoparticle formulations of the mRNAsdescribed herein may include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or8) cationic and/or ionizable lipids. Such cationic and/or ionizablelipids include, but are not limited to,3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10),N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine(KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25),1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA),2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA),heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate(DLin-MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane(DLin-KC2-DMA),2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine(Octyl-CLinDMA),(2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine(Octyl-CLinDMA (2R)),(2S)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine(Octyl-CLinDMA (2S)).N,N-dioleyl-N,N-dimethylammonium chloride(“DODAC”); N-(2,3-dioleyloxy)propyl-N,N—N-triethylammonium chloride(“DOTMA”); N,N-distearyl-N,N-dimethylammonium bromide (“DDAB”);N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (“DOTAP”);1,2-Dioleyloxy-3-trimethylaminopropane chloride salt (“DOTAP.Cl”);3-β-(N—(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (“DC-Chol”),N-(1-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethyl-ammoniumtrifluoracetate (“DOSPA”), dioctadecylamidoglycyl carboxyspermine(“DOGS”), 1,2-dioleoyl-3-dimethylammonium propane (“DODAP”),N,N-dimethyl-2,3-dioleyloxy)propylamine (“DODMA”), andN-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (“DMRIE”). Additionally, a number of commercial preparations ofcationic and/or ionizable lipids can be used, such as, e.g., LIPOFECTIN®(including DOTMA and DOPE, available from GIBCO/BRL), and LIPOFECTAMINE®(including DOSPA and DOPE, available from GIBCO/BRL). KL10, KL22, andKL25 are described, for example, in U.S. Pat. No. 8,691,750, which isincorporated herein by reference in its entirety. In particularembodiments, the lipid is DLin-MC3-DMA or DLin-KC2-DMA.

Anionic lipids suitable for use in lipid nanoparticles of the disclosureinclude, but are not limited to, phosphatidylglycerol, cardiolipin,diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanoloamine, N-succinyl phosphatidylethanolamine,N-glutaryl phosphatidylethanolamine, lysylphosphatidylglycerol, andother anionic modifying groups joined to neutral lipids.

Neutral lipids suitable for use in lipid nanoparticles of the disclosureinclude, but are not limited to, diacylphosphatidylcholine,diacylphosphatidylethanolamine, ceramide, sphingomyelin,dihydrosphingomyelin, cephalin, and cerebrosides. Lipids having avariety of acyl chain groups of varying chain length and degree ofsaturation are available or may be isolated or synthesized by well-knowntechniques. Additionally, lipids having mixtures of saturated andunsaturated fatty acid chains can be used. In some embodiments, theneutral lipids used in the disclosure are DOPE, DSPC, DPPC, POPC, or anyrelated phosphatidylcholine. In some embodiments, the neutral lipid maybe composed of sphingomyelin, dihydrosphingomyeline, or phospholipidswith other head groups, such as serine and inositol.

In some embodiments, amphipathic lipids are included in nanoparticles ofthe disclosure. Exemplary amphipathic lipids suitable for use innanoparticles of the disclosure include, but are not limited to,sphingolipids, phospholipids, and aminolipids. In some embodiments, aphospholipid is selected from the group consisting of1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC),1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC),1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC),1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC),1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine(OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC),1,2-dilinolenoyl-sn-glycero-3-phosphocholine,1,2-diarachidonoyl-sn-glycero-3-phosphocholine,1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine,1,2-dioleoyl-sn-glycero-3-phosphoetha nolamine (DOPE),1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE),1,2-distearoyl-sn-glycero-3-phosphoethanolamine,1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine,1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine,1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine,1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine,1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG),and sphingomyelin. Other phosphorus-lacking compounds, such assphingolipids, glycosphingolipid families, diacylglycerols, andβ-acyloxyacids, may also be used. Additionally, such amphipathic lipidscan be readily mixed with other lipids, such as triglycerides andsterols.

In some embodiments, the lipid component of a nanoparticle of thedisclosure may include one or more PEGylated lipids. A PEGylated lipid(also known as a PEG lipid or a PEG-modified lipid) is a lipid modifiedwith polyethylene glycol. The lipid component may include one or morePEGylated lipids. A PEGylated lipid may be selected from thenon-limiting group consisting of PEG-modified phosphatidylethanolamines,PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modifieddialkylamines, PEG-modified diacylglycerols, and PEG-modifieddialkylglycerols. For example, a PEGylated lipid may be PEG-c-DOMG,PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.

A lipid nanoparticle of the disclosure may include one or morestructural lipids. Exemplary, non-limiting structural lipids that may bepresent in the lipid nanoparticles of the disclosure includecholesterol, fecosterol, sitosterol, campesterol, stigmasterol,brassicasterol, ergosterol, tomatidine, tomatine, ursolic acid, oralpha-tocopherol.

In some embodiments, one or more mRNA of the disclosure may beformulated in a lipid nanoparticle having a diameter from about 1 nm toabout 900 nm, e.g., about 1 nm to about 100 nm, about 1 nm to about 200nm, about 1 nm to about 300 nm, about 1 nm to about 400 nm, about 1 nmto about 500 nm, about 1 nm to about 600 nm, about 1 nm to about 700 nm,about 1 nm to 800 nm, about 1 nm to about 900 nm. In some embodiments,the nanoparticle may have a diameter from about 10 nm to about 300 nm,about 20 nm to about 200 nm, about 30 nm to about 100 nm, or about 40 nmto about 80 nm. In some embodiments, the nanoparticle may have adiameter from about 30 nm to about 300 nm, about 40 nm to about 200 nm,about 50 nm to about 150 nm, about 70 to about 110 nm, or about 80 nm toabout 120 nm. In one embodiment, an mRNA may be formulated in a lipidnanoparticle having a diameter from about 10 to about 100 nm includingranges in between such as, but not limited to, about 10 to about 20 nm,about 10 to about 30 nm, about 10 to about 40 nm, about 10 to about 50nm, about 10 to about 60 nm, about 10 to about 70 nm, about 10 to about80 nm, about 10 to about 90 nm, about 20 to about 30 nm, about 20 toabout 40 nm, about 20 to about 50 nm, about 20 to about 60 nm, about 20to about 70 nm, about 20 to about 80 nm, about 20 to about 90 nm, about20 to about 100 nm, about 30 to about 40 nm, about 30 to about 50 nm,about 30 to about 60 nm, about 30 to about 70 nm, about 30 to about 80nm, about 30 to about 90 nm, about 30 to about 100 nm, about 40 to about50 nm, about 40 to about 60 nm, about 40 to about 70 nm, about 40 toabout 80 nm, about 40 to about 90 nm, about 40 to about 100 nm, about 50to about 60 nm, about 50 to about 70 nm about 50 to about 80 nm, about50 to about 90 nm, about 50 to about 100 nm, about 60 to about 70 nm,about 60 to about 80 nm, about 60 to about 90 nm, about 60 to about 100nm, about 70 to about 80 nm, about 70 to about 90 nm, about 70 to about100 nm, about 80 to about 90 nm, about 80 to about 100 nm, and/or about90 to about 100 nm. In one embodiment, an mRNA may be formulated in alipid nanoparticle having a diameter from about 30 nm to about 300 nm,about 40 nm to about 200 nm, about 50 nm to about 150 nm, about 70 toabout 110 nm, or about 80 nm to about 120 nm including ranges inbetween.

In some embodiments, a lipid nanoparticle may have a diameter greaterthan 100 nm, greater than 150 nm, greater than 200 nm, greater than 250nm, greater than 300 nm, greater than 350 nm, greater than 400 nm,greater than 450 nm, greater than 500 nm, greater than 550 nm, greaterthan 600 nm, greater than 650 nm, greater than 700 nm, greater than 750nm, greater than 800 nm, greater than 850 nm, greater than 900 nm, orgreater than 950 nm.

In some embodiments, the particle size of the lipid nanoparticle may beincreased and/or decreased. The change in particle size may be able tohelp counter a biological reaction such as, but not limited to,inflammation, or may increase the biological effect of the mRNAdelivered to a patient or subject.

In certain embodiments, it is desirable to target a nanoparticle, e.g.,a lipid nanoparticle, of the disclosure using a targeting moiety that isspecific to a cell type and/or tissue type. In some embodiments, ananoparticle may be targeted to a particular cell, tissue, and/or organusing a targeting moiety. In particular embodiments, a nanoparticlecomprises one or more mRNA described herein and a targeting moiety.Exemplary non-limiting targeting moieties include ligands, cell surfacereceptors, glycoproteins, vitamins (e.g., riboflavin) and antibodies(e.g., full-length antibodies, antibody fragments (e.g., Fv fragments,single chain Fv (scFv) fragments, Fab′ fragments, or F(ab′)2 fragments),single domain antibodies, camelid antibodies and fragments thereof,human antibodies and fragments thereof, monoclonal antibodies, andmultispecific antibodies (e.g., bispecific antibodies)). In someembodiments, the targeting moiety may be a polypeptide. The targetingmoiety may include the entire polypeptide (e.g., peptide or protein) orfragments thereof. A targeting moiety is typically positioned on theouter surface of the nanoparticle in such a manner that the targetingmoiety is available for interaction with the target, for example, a cellsurface receptor. A variety of different targeting moieties and methodsare known and available in the art, including those described, e.g., inSapra et al., Prog. Lipid Res. 42(5):439-62, 2003 and Abra et al., J.Liposome Res. 12:1-3, 2002.

In some embodiments, a lipid nanoparticle (e.g., a liposome) may includea surface coating of hydrophilic polymer chains, such as polyethyleneglycol (PEG) chains (see, e.g., Allen et al., Biochimica et BiophysicaActa 1237: 99-108, 1995; DeFrees et al., Journal of the AmericanChemistry Society 118: 6101-6104, 1996; Blume et al., Biochimica etBiophysica Acta 1149: 180-184, 1993; Klibanov et al., Journal ofLiposome Research 2: 321-334, 1992; U.S. Pat. No. 5,013,556; Zalipsky,Bioconjugate Chemistry 4: 296-299, 1993; Zalipsky, FEBS Letters 353:71-74, 1994; Zalipsky, in Stealth Liposomes Chapter 9 (Lasic and Martin,Eds) CRC Press, Boca Raton Fla., 1995). In one approach, a targetingmoiety for targeting the lipid nanoparticle is linked to the polar headgroup of lipids forming the nanoparticle. In another approach, thetargeting moiety is attached to the distal ends of the PEG chainsforming the hydrophilic polymer coating (see, e.g., Klibanov et al.,Journal of Liposome Research 2: 321-334, 1992; Kirpotin et al., FEBSLetters 388: 115-118, 1996).

Standard methods for coupling the targeting moiety or moieties may beused. For example, phosphatidylethanolamine, which can be activated forattachment of targeting moieties, or derivatized lipophilic compounds,such as lipid-derivatized bleomycin, can be used. Antibody-targetedliposomes can be constructed using, for instance, liposomes thatincorporate protein A (see, e.g., Renneisen et al., J. Bio. Chem.,265:16337-16342, 1990 and Leonetti et al., Proc. Natl. Acad. Sci. (USA),87:2448-2451, 1990). Other examples of antibody conjugation aredisclosed in U.S. Pat. No. 6,027,726. Examples of targeting moieties canalso include other polypeptides that are specific to cellularcomponents, including antigens associated with neoplasms or tumors.Polypeptides used as targeting moieties can be attached to the liposomesvia covalent bonds (see, for example Heath, Covalent Attachment ofProteins to Liposomes, 149 Methods in Enzymology 111-119 (AcademicPress, Inc. 1987)). Other targeting methods include the biotin-avidinsystem.

In some embodiments, a lipid nanoparticle of the disclosure includes atargeting moiety that targets the lipid nanoparticle to a cellincluding, but not limited to, hepatocytes, colon cells, epithelialcells, hematopoietic cells, epithelial cells, endothelial cells, lungcells, bone cells, stem cells, mesenchymal cells, neural cells, cardiaccells, adipocytes, vascular smooth muscle cells, cardiomyocytes,skeletal muscle cells, beta cells, pituitary cells, synovial liningcells, ovarian cells, testicular cells, fibroblasts, B cells, T cells,reticulocytes, leukocytes, granulocytes, and tumor cells (includingprimary tumor cells and metastatic tumor cells). In particularembodiments, the targeting moiety targets the lipid nanoparticle to ahepatocyte. In other embodiments, the targeting moiety targets the lipidnanoparticle to a colon cell. In some embodiments, the targeting moietytargets the lipid nanoparticle to a liver cancer cell (e.g., ahepatocellular carcinoma cell) or a colorectal cancer cell (e.g., aprimary tumor or a metastasis).

Lipid Nanoparticles

In one set of embodiments, lipid nanoparticles (LNPs) are provided. Inone embodiment, a lipid nanoparticle comprises lipids including anionizable lipid, a structural lipid, a phospholipid, and one or moremRNAs. Each of the LNPs described herein may be used as a formulationfor the mRNA described herein. In one embodiment, a lipid nanoparticlecomprises an ionizable lipid, a structural lipid, a phospholipid, aPEG-modified lipid and one or more mRNAs. In some embodiments, the LNPcomprises an ionizable lipid, a PEG-modified lipid, a sterol and aphospholipid. In some embodiments, the LNP has a molar ratio of about20-60% ionizable lipid:about 5-25% phospholipid:about 25-55% sterol; andabout 0.5-15% PEG-modified lipid. In some embodiments, the LNP comprisesa molar ratio of about 50% ionizable lipid, about 1.5% PEG-modifiedlipid, about 38.5% cholesterol and about 10% phospholipid. In someembodiments, the LNP comprises a molar ratio of about 55% ionizablelipid, about 2.5% PEG lipid, about 32.5% cholesterol and about 10%phospholipid. In some embodiments, the ionizable lipid is an ionizableamino or cationic lipid and the neutral lipid is a phospholipid, and thesterol is a cholesterol. In some embodiments, the LNP has a molar ratioof 50:38.5:10:1.5 of ionizable lipid:cholesterol:DSPC(1,2-dioctadecanoyl-sn-glycero-3-phosphocholine):PEG-DMG.

a. Ionizable Lipid

The present disclosure provides pharmaceutical compositions withadvantageous properties. For example, the lipids described herein (e.g.those having any of Formula (I), (IA), (II), (IIa), (IIb), (IIc), (IId),(IIe), (III), (IV), (V), or (VI) may be advantageously used in lipidnanoparticle compositions for the delivery of therapeutic and/orprophylactic agents to mammalian cells or organs. For example, thelipids described herein have little or no immunogenicity. For example,the lipid compounds disclosed hereinhave a lower immunogenicity ascompared to a reference lipid (e.g., MC3, KC2, or DLinDMA). For example,a formulation comprising a lipid disclosed herein and a therapeutic orprophylactic agent has an increased therapeutic index as compared to acorresponding formulation which comprises a reference lipid (e.g., MC3,KC2, or DLinDMA) and the same therapeutic or prophylactic agent. Inparticular, the present application provides pharmaceutical compositionscomprising:

(a) a polynucleotide comprising a nucleotide sequence encoding apolypeptide of interest; and

(b) a delivery agent.

In some embodiments, the delivery agent comprises a lipid compoundhaving the Formula (I)

wherein

R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;

R₂ and R₃ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;

R₄ is selected from the group consisting of a C₃₋₆ carbocycle,—(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted C₁₋₆alkyl, where Q is selected from a carbocycle, heterocycle, —OR,—O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN, —N(R)₂,—C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂,—N(R)R₈, —O(CH₂)_(n)OR, —N(R)C(═NR₉)N(R)₂, —N(R)C(═CHR₉)N(R)₂,—OC(O)N(R)₂, —N(R)C(O)OR, —N(OR)C(O)R, —N(OR)S(O)₂R, —N(OR)C(O)OR,—N(OR)C(O)N(R)₂, —N(OR)C(S)N(R)₂, —N(OR)C(═NR₉)N(R)₂,—N(OR)C(═CHR₉)N(R)₂, —C(═NR₉)N(R)₂, —C(═NR₉)R, —C(O)N(R)OR, and—C(R)N(R)₂C(O)OR, and each n is independently selected from 1, 2, 3, 4,and 5;

each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group;

R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

R₈ is selected from the group consisting of C₃₋₆ carbocycle andheterocycle;

R₉ is selected from the group consisting of H, CN, NO₂, C₁₋₆ alkyl, —OR,—S(O)₂R, —S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆ carbocycle and heterocycle;

each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;

each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13, orsalts or stereoisomers thereof.

In some embodiments, a subset of compounds of Formula (I) includes thosein which

R₁ is selected from the group consisting of C₅₋₂₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;

R₂ and R₃ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;

R₄ is selected from the group consisting of a C₃₋₆ carbocycle,—(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted C₁₋₆alkyl, where Q is selected from a carbocycle, heterocycle, —OR,—O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN, —N(R)₂,—C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂, and—C(R)N(R)₂C(O)OR, and each n is independently selected from 1, 2, 3, 4,and 5;

each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, an aryl group, and a heteroaryl group;

R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;

each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,

or salts or stereoisomers thereof, wherein alkyl and alkenyl groups maybe linear or branched.

In some embodiments, a subset of compounds of Formula (I) includes thosein which when R₄ is —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, or —CQ(R)₂, then(i) Q is not —N(R)₂ when n is 1, 2, 3, 4 or 5, or (ii) Q is not 5, 6, or7-membered heterocycloalkyl when n is 1 or 2.

In another embodiments, another subset of compounds of Formula (I)includes those in which

R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;

R₂ and R₃ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;

R₄ is selected from the group consisting of a C₃₋₆ carbocycle,—(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted C₁₋₆alkyl, where Q is selected from a C₃₋₆ carbocycle, a 5- to 14-memberedheteroaryl having one or more heteroatoms selected from N, O, and S,—OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN,—C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂,—CRN(R)₂C(O)OR, —N(R)R₈, —O(CH₂)_(n)OR, —N(R)C(═NR₉)N(R)₂,—N(R)C(═CHR₉)N(R)₂, —O C(O)N(R)₂, —N(R)C(O)OR, —N(OR)C(O)R,—N(OR)S(O)₂R, —N(OR)C(O)OR, —N(OR)C(O)N(R)₂, —N(OR)C(S)N(R)₂,—N(OR)C(═NR₉)N(R)₂, —N(OR)C(═CHR₉)N(R)₂, —C(═NR₉)N(R)₂, —C (═NR₉)R,—C(O)N(R)OR, and a 5- to 14-membered heterocycloalkyl having one or moreheteroatoms selected from N, O, and S which is substituted with one ormore substituents selected from oxo (═O), OH, amino, and C₁₋₃ alkyl, andeach n is independently selected from 1, 2, 3, 4, and 5;

each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group;

R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

R₈ is selected from the group consisting of C₃₋₆ carbocycle andheterocycle;

R₉ is selected from the group consisting of H, CN, NO₂, C₁₋₆ alkyl, —OR,—S(O)₂R, —S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆ carbocycle and heterocycle;

each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;

each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,

or salts or stereoisomers thereof.

In another embodiments, another subset of compounds of Formula (I)includes those in which

R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;

R₂ and R₃ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;

R₄ is selected from the group consisting of a C₃₋₆ carbocycle,—(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted C₁₋₆alkyl, where Q is selected from a C₃₋₆ carbocycle, a 5- to 14-memberedheteroaryl having one or more heteroatoms selected from N, O, and S,—OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN,—C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂,—CRN(R)₂C(O)OR, and a 5- to 14-membered heterocycloalkyl having one ormore heteroatoms selected from N, O, and S which is substituted with oneor more substituents selected from oxo (═O), OH, amino, and C₁₋₃ alkyl,and each n is independently selected from 1, 2, 3, 4, and 5;

each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, an aryl group, and a heteroaryl group;

R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;

each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,

or salts or stereoisomers thereof.

In yet another embodiments, another subset of compounds of Formula (I)includes those in which

R₁ is selected from the group consisting of C₅₋₂₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;

R₂ and R₃ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;

R₄ is selected from the group consisting of a C₃₋₆ carbocycle,—(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted C₁₋₆alkyl, where Q is selected from a C₃₋₆ carbocycle, a 5- to 14-memberedheterocycle having one or more heteroatoms selected from N, O, and S,—OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN,—C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂,—CRN(R)₂C(O)OR, —N(R)R₈, —O(CH₂)_(n)OR, —N(R)C(═NR₉)N(R)₂,—N(R)C(═CHR₉)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR, —N(OR)C(O)R, —N(OR)S(O)₂R,—N(OR)C(O)OR, —N(OR)C(O)N(R)₂, —N(OR)C(S)N(R)₂, —N(OR)C(═NR₉)N(R)₂,—N(OR)C(═CHR₉)N(R)₂, —C(═NR₉)R, —C(O)N(R)OR, and —C(═NR₉)N(R)₂, and eachn is independently selected from 1, 2, 3, 4, and 5; and when Q is a 5-to 14-membered heterocycle and (i) R₄ is —(CH₂)_(n)Q in which n is 1 or2, or (ii) R₄ is —(CH₂)_(n)CHQR in which n is 1, or (iii) R₄ is —CHQR,and —CQ(R)₂, then Q is either a 5- to 14-membered heteroaryl or 8- to14-membered heterocycloalkyl;

each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group;

R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

R₈ is selected from the group consisting of C₃₋₆ carbocycle andheterocycle;

R₉ is selected from the group consisting of H, CN, NO₂, C₁₋₆ alkyl, —OR,—S(O)₂R, —S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆ carbocycle and heterocycle;

each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;

each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,

or salts or stereoisomers thereof.

In yet another embodiments, another subset of compounds of Formula (I)includes those in which

R₁ is selected from the group consisting of C₅₋₂₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;

R₂ and R₃ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;

R₄ is selected from the group consisting of a C₃₋₆ carbocycle,—(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted C₁₋₆alkyl, where Q is selected from a C₃₋₆ carbocycle, a 5- to 14-memberedheterocycle having one or more heteroatoms selected from N, O, and S,—OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN,—C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂,—CRN(R)₂C(O)OR, and each n is independently selected from 1, 2, 3, 4,and 5; and when Q is a 5- to 14-membered heterocycle and (i) R₄ is—(CH₂)_(n)Q in which n is 1 or 2, or (ii) R₄ is —(CH₂)_(n)CHQR in whichn is 1, or (iii) R₄ is —CHQR, and —CQ(R)₂, then Q is either a 5- to14-membered heteroaryl or 8- to 14-membered heterocycloalkyl;

each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, an aryl group, and a heteroaryl group;

R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;

each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,

or salts or stereoisomers thereof.

In still another embodiments, another subset of compounds of Formula (I)includes those in which

R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;

R₂ and R₃ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;

R₄ is selected from the group consisting of a C₃₋₆ carbocycle,—(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted C₁₋₆alkyl, where Q is selected from a C₃₋₆ carbocycle, a 5- to 14-memberedheteroaryl having one or more heteroatoms selected from N, O, and S,—OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN,—C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂,—CRN(R)₂C(O)OR, —N(R)R₈, —O(CH₂)_(n)OR, —N(R)C(═NR₉)N(R)₂,—N(R)C(═CHR₉)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR, —N(OR)C(O)R, —N(OR)S(O)₂R,—N(OR)C(O)OR, —N(OR)C(O)N(R)₂, —N(OR)C(S)N(R)₂, —N(OR)C(═NR₉)N(R)₂,—N(OR)C(═CHR₉)N(R)₂, —C(═NR₉)R, —C(O)N(R)OR, and —C(═NR₉)N(R)₂, and eachn is independently selected from 1, 2, 3, 4, and 5;

each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group;

R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

R₈ is selected from the group consisting of C₃₋₆ carbocycle andheterocycle;

R₉ is selected from the group consisting of H, CN, NO₂, C₁₋₆ alkyl, —OR,—S(O)₂R, —S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆ carbocycle and heterocycle;

each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;

each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,

or salts or stereoisomers thereof.

In still another embodiments, another subset of compounds of Formula (I)includes those in which

R₁ is selected from the group consisting of C₅₋₂₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;

R₂ and R₃ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;

R₄ is selected from the group consisting of a C₃₋₆ carbocycle,—(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted C₁₋₆alkyl, where Q is selected from a C₃₋₆ carbocycle, a 5- to 14-memberedheteroaryl having one or more heteroatoms selected from N, O, and S,—OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN,—C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂,—CRN(R)₂C(O)OR, and each n is independently selected from 1, 2, 3, 4,and 5;

each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, an aryl group, and a heteroaryl group;

R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;

each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,

or salts or stereoisomers thereof.

In yet another embodiments, another subset of compounds of Formula (I)includes those in which

R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;

R₂ and R₃ are independently selected from the group consisting of H,C₂₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;

R₄ is —(CH₂)_(n)Q or —(CH₂)_(n)CHQR, where Q is —N(R)₂, and n isselected from 3, 4, and 5;

each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group;

R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;

each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₁₋₁₂ alkenyl;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,

or salts or stereoisomers thereof.

In yet another embodiments, another subset of compounds of Formula (I)includes those in which

R₁ is selected from the group consisting of C₅₋₂₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;

R₂ and R₃ are independently selected from the group consisting of H,C₂₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;

R₄ is —(CH₂)_(n)Q or —(CH₂)_(n)CHQR, where Q is —N(R)₂, and n isselected from 3, 4, and 5;

each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, an aryl group, and a heteroaryl group;

R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;

each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₁₋₁₂ alkenyl;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,

or salts or stereoisomers thereof.

In still other embodiments, another subset of compounds of Formula (I)includes those in which

R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;

R₂ and R₃ are independently selected from the group consisting of C₁₋₁₄alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃, togetherwith the atom to which they are attached, form a heterocycle orcarbocycle;

R₄ is selected from the group consisting of —(CH₂)_(n)Q, —(CH₂)_(n)CHQR,—CHQR, and —CQ(R)₂, where Q is —N(R)₂, and n is selected from 1, 2, 3,4, and 5;

each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group;

R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;

each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₁₋₁₂ alkenyl;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,

or salts or stereoisomers thereof.

In still other embodiments, another subset of compounds of Formula (I)includes those in which

R₁ is selected from the group consisting of C₅₋₂₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;

R₂ and R₃ are independently selected from the group consisting of C₁₋₁₄alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃, togetherwith the atom to which they are attached, form a heterocycle orcarbocycle;

R₄ is selected from the group consisting of —(CH₂)_(n)Q, —(CH₂)_(n)CHQR,—CHQR, and —CQ(R)₂, where Q is —N(R)₂, and n is selected from 1, 2, 3,4, and 5;

each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, an aryl group, and a heteroaryl group;

R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;

each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₁₋₁₂ alkenyl;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,

or salts or stereoisomers thereof.

In certain embodiments, a subset of compounds of Formula (I) includesthose of Formula (IA):

or a salt or stereoisomer thereof, wherein 1 is selected from 1, 2, 3,4, and 5; m is selected from 5, 6, 7, 8, and 9; M₁ is a bond or M′; R₄is unsubstituted C₁₋₃ alkyl, or —(CH₂)_(n)Q, in which Q is OH,—NHC(S)N(R)₂, —NHC(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)R₈,—NHC(═NR₉)N(R)₂, —NHC(═CHR₉)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR, heteroaryl,or heterocycloalkyl; M and M′ are independently selected from —C(O)O—,—OC(O)—, —C(O)N(R′)—, —P(O)(OR′)O—, —S—S—, an aryl group, and aheteroaryl group; and

R₂ and R₃ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, and C₂₋₁₄ alkenyl.

In some embodiments, a subset of compounds of Formula (I) includes thoseof Formula (IA), or a salt or stereoisomer thereof,

wherein

1 is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, and9;

M₁ is a bond or M′;

R₄ is unsubstituted C₁₋₃ alkyl, or —(CH₂)_(n)Q, in which Q is OH,—NHC(S)N(R)₂, or —NHC(O)N(R)₂;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—P(O)(OR′)O—, an aryl group, and a heteroaryl group; and

R₂ and R₃ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, and C₂₋₁₄ alkenyl.

In certain embodiments, a subset of compounds of Formula (I) includesthose of Formula (II):

or a salt or stereoisomer thereof, wherein 1 is selected from 1, 2, 3,4, and 5; M₁ is a bond or M′; R₄ is unsubstituted C₁₋₃ alkyl, or—(CH₂)_(n)Q, in which n is 2, 3, or 4, and Q is OH, —NHC(S)N(R)₂,—NHC(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)R₈, —NHC(═NR₉)N(R)₂,—NHC(═CHR₉)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR, heteroaryl, orheterocycloalkyl; M and M′ are independently selected from —C(O)O—,—OC(O)—, —C(O)N(R′)—, —P(O)(OR′)O—, —S—S—, an aryl group, and aheteroaryl group; and

R₂ and R₃ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, and C₂₋₁₄ alkenyl.

In some embodiments, a subset of compounds of Formula (I) includes thoseof Formula (II), or a salt or stereoisomer thereof, wherein

1 is selected from 1, 2, 3, 4, and 5;

M₁ is a bond or M′;

R₄ is unsubstituted C₁₋₃ alkyl, or —(CH₂)_(n)Q, in which n is 2, 3, or4, and Q is OH, —NHC(S)N(R)₂, or —NHC(O)N(R)₂;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—P(O)(OR′)O—, an aryl group, and a heteroaryl group; and

R₂ and R₃ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, and C₂₋₁₄ alkenyl.

In some embodiments, the compound of formula (I) is of the formula(IIa),

or a salt thereof, wherein R₄ is as described above.

In some embodiments, the compound of formula (I) is of the formula(IIb),

or a salt thereof, wherein R₄ is as described above.

In some embodiments, the compound of formula (I) is of the formula(IIc),

or a salt thereof, wherein R₄ is as described above.

In some embodiments, the compound of formula (I) is of the formula(IIe):

or a salt thereof, wherein R₄ is as described above.

In some embodiments, the compound of formula (IIa), (IIb), (IIc), or(IIe) comprises an R₄ which is selected from —(CH₂)_(n)Q and—(CH₂)_(n)CHQR, wherein Q, R and n are as defined above.

In some embodiments, Q is selected from the group consisting of —OR,—OH, —O(CH₂)_(n)N(R)₂, —OC(O)R, —CX₃, —CN, —N(R)C(O)R, —N(H)C(O)R,—N(R)S(O)₂R, —N(H)S(O)₂R, —N(R)C(O)N(R)₂, —N(H)C(O)N(R)₂,—N(H)C(O)N(H)(R), —N(R)C(S)N(R)₂, —N(H)C(S)N(R)₂, —N(H)C(S)N(H)(R), anda heterocycle, wherein R is as defined above. In some aspects, n is 1 or2. In some embodiments, Q is OH, —NHC(S)N(R)₂, or —NHC(O)N(R)₂.

In some embodiments, the compound of formula (I) is of the formula(IId),

or a salt thereof, wherein R₂ and R₃ are independently selected from thegroup consisting of C₅₋₁₄ alkyl and C₅₋₁₄ alkenyl, n is selected from 2,3, and 4, and R′, R″, R₅, R₆ and m are as defined above.

In some aspects of the compound of formula (IId), R₂ is C₈ alkyl. Insome aspects of the compound of formula (IId), R₃ is C₅-C₉ alkyl. Insome aspects of the compound of formula (IId), m is 5, 7, or 9. In someaspects of the compound of formula (IId), each R₅ is H. In some aspectsof the compound of formula (IId), each R₆ is H.

In another aspect, the present application provides a lipid composition(e.g., a lipid nanoparticle (LNP)) comprising: (1) a compound having theformula (I); (2) optionally a helper lipid (e.g. a phospholipid); (3)optionally a structural lipid (e.g. a sterol); and (4) optionally alipid conjugate (e.g. a PEG-lipid). In exemplary embodiments, the lipidcomposition (e.g., LNP) further comprises a polynucleotide encoding apolypeptide of interest, e.g., a polynucleotide encapsulated therein.

As used herein, the term “alkyl” or “alkyl group” means a linear orbranched, saturated hydrocarbon including one or more carbon atoms(e.g., one, two, three, four, five, six, seven, eight, nine, ten,eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen,eighteen, nineteen, twenty, or more carbon atoms).

The notation “C₁₋₁₄ alkyl” means a linear or branched, saturatedhydrocarbon including 1-14 carbon atoms. An alkyl group can beoptionally substituted.

As used herein, the term “alkenyl” or “alkenyl group” means a linear orbranched hydrocarbon including two or more carbon atoms (e.g., two,three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen,twenty, or more carbon atoms) and at least one double bond.

The notation “C₂₋₁₄ alkenyl” means a linear or branched hydrocarbonincluding 2-14 carbon atoms and at least one double bond. An alkenylgroup can include one, two, three, four, or more double bonds. Forexample, C₁₈ alkenyl can include one or more double bonds. A C₁₈ alkenylgroup including two double bonds can be a linoleyl group. An alkenylgroup can be optionally substituted.

As used herein, the term “carbocycle” or “carbocyclic group” means amono- or multi-cyclic system including one or more rings of carbonatoms. Rings can be three, four, five, six, seven, eight, nine, ten,eleven, twelve, thirteen, fourteen, or fifteen membered rings.

The notation “C₃₋₆ carbocycle” means a carbocycle including a singlering having 3-6 carbon atoms. Carbocycles can include one or more doublebonds and can be aromatic (e.g., aryl groups). Examples of carbocyclesinclude cyclopropyl, cyclopentyl, cyclohexyl, phenyl, naphthyl, and1,2-dihydronaphthyl groups. Carbocycles can be optionally substituted.

As used herein, the term “heterocycle” or “heterocyclic group” means amono- or multi-cyclic system including one or more rings, where at leastone ring includes at least one heteroatom. Heteroatoms can be, forexample, nitrogen, oxygen, or sulfur atoms. Rings can be three, four,five, six, seven, eight, nine, ten, eleven, or twelve membered rings.Heterocycles can include one or more double bonds and can be aromatic(e.g., heteroaryl groups). Examples of heterocycles include imidazolyl,imidazolidinyl, oxazolyl, oxazolidinyl, thiazolyl, thiazolidinyl,pyrazolidinyl, pyrazolyl, isoxazolidinyl, isoxazolyl, isothiazolidinyl,isothiazolyl, morpholinyl, pyrrolyl, pyrrolidinyl, furyl,tetrahydrofuryl, thiophenyl, pyridinyl, piperidinyl, quinolyl, andisoquinolyl groups. Heterocycles can be optionally substituted.

As used herein, a “biodegradable group” is a group that can facilitatefaster metabolism of a lipid in a subject. A biodegradable group can be,but is not limited to, —C(O)O—, —OC(O)—, —C(O)N(R′)—, —N(R′)C(O)—,—C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O)₂—, anaryl group, and a heteroaryl group.

As used herein, an “aryl group” is a carbocyclic group including one ormore aromatic rings. Examples of aryl groups include phenyl and naphthylgroups.

As used herein, a “heteroaryl group” is a heterocyclic group includingone or more aromatic rings. Examples of heteroaryl groups includepyrrolyl, furyl, thiophenyl, imidazolyl, oxazolyl, and thiazolyl. Botharyl and heteroaryl groups can be optionally substituted. For example, Mand M′ can be selected from the non-limiting group consisting ofoptionally substituted phenyl, oxazole, and thiazole. In the formulasherein, M and M′ can be independently selected from the list ofbiodegradable groups above.

Alkyl, alkenyl, and cyclyl (e.g., carbocyclyl and heterocyclyl) groupscan be optionally substituted unless otherwise specified. Optionalsubstituents can be selected from the group consisting of, but are notlimited to, a halogen atom (e.g., a chloride, bromide, fluoride, oriodide group), a carboxylic acid (e.g., —C(O)OH), an alcohol (e.g., ahydroxyl, —OH), an ester (e.g., —C(O)OR or —OC(O)R), an aldehyde (e.g.,—C(O)H), a carbonyl (e.g., —C(O)R, alternatively represented by C═O), anacyl halide (e.g., —C(O)X, in which X is a halide selected from bromide,fluoride, chloride, and iodide), a carbonate (e.g., —OC(O)OR), an alkoxy(e.g., —OR), an acetal (e.g., —C(OR)₂R″″, in which each OR are alkoxygroups that can be the same or different and R″″ is an alkyl or alkenylgroup), a phosphate (e.g., P(O)₄ ³⁻), a thiol (e.g., —SH), a sulfoxide(e.g., —S(O)R), a sulfinic acid (e.g., —S(O)OH), a sulfonic acid (e.g.,—S(O)₂OH), a thial (e.g., —C(S)H), a sulfate (e.g., S(O)₄ ²⁻), asulfonyl (e.g., —S(O)₂—), an amide (e.g., —C(O)NR₂, or —N(R)C(O)R), anazido (e.g., —N₃), a nitro (e.g., —NO₂), a cyano (e.g., —CN), anisocyano (e.g., —NC), an acyloxy (e.g., —OC(O)R), an amino (e.g., —NR₂,—NRH, or —NH₂), a carbamoyl (e.g., —OC(O)NR₂, —OC(O)NRH, or —OC(O)NH₂),a sulfonamide (e.g., —S(O)₂NR₂, —S(O)₂NRH, —S(O)₂NH₂, —N(R)S(O)₂R,—N(H)S(O)₂R, —N(R)S(O)₂H, or —N(H)S(O)₂H), an alkyl group, an alkenylgroup, and a cyclyl (e.g., carbocyclyl or heterocyclyl) group.

In any of the preceding, R is an alkyl or alkenyl group, as definedherein. In some embodiments, the substituent groups themselves can befurther substituted with, for example, one, two, three, four, five, orsix substituents as defined herein. For example, a C₁₋₆ alkyl group canbe further substituted with one, two, three, four, five, or sixsubstituents as described herein.

The compounds of any one of formulae (I), (IA), (II), (IIa), (IIb),(IIc), (IId), and (IIe) include one or more of the following featureswhen applicable.

In some embodiments, R₄ is selected from the group consisting of a C₃₋₆carbocycle, —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, and —CQ(R)₂, where Q isselected from a C₃₋₆ carbocycle, 5- to 14-membered aromatic ornon-aromatic heterocycle having one or more heteroatoms selected from N,O, S, and P, —OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H,—CXH₂, —CN, —N(R)₂, —C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂,—N(R)C(S)N(R)₂, and —C(R)N(R)₂C(O)OR, and each n is independentlyselected from 1, 2, 3, 4, and 5.

In another embodiment, R₄ is selected from the group consisting of aC₃₋₆ carbocycle, —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, and —CQ(R)₂, whereQ is selected from a C₃₋₆ carbocycle, a 5- to 14-membered heteroarylhaving one or more heteroatoms selected from N, O, and S, —OR,—O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN, —C(O)N(R)₂,—N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂,—C(R)N(R)₂C(O)OR, and a 5- to 14-membered heterocycloalkyl having one ormore heteroatoms selected from N, O, and S which is substituted with oneor more substituents selected from oxo (═O), OH, amino, and C₁₋₃ alkyl,and each n is independently selected from 1, 2, 3, 4, and 5.

In another embodiment, R₄ is selected from the group consisting of aC₃₋₆ carbocycle, —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, and —CQ(R)₂, whereQ is selected from a C₃₋₆ carbocycle, a 5- to 14-membered heterocyclehaving one or more heteroatoms selected from N, O, and S, —OR,—O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN, —C(O)N(R)₂,—N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂,—C(R)N(R)₂C(O)OR, and each n is independently selected from 1, 2, 3, 4,and 5; and when Q is a 5- to 14-membered heterocycle and (i) R₄ is—(CH₂)_(n)Q in which n is 1 or 2, or (ii) R₄ is —(CH₂)_(n)CHQR in whichn is 1, or (iii) R₄ is —CHQR, and —CQ(R)₂, then Q is either a 5- to14-membered heteroaryl or 8- to 14-membered heterocycloalkyl.

In another embodiment, R₄ is selected from the group consisting of aC₃₋₆ carbocycle, —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, and —CQ(R)₂, whereQ is selected from a C₃₋₆ carbocycle, a 5- to 14-membered heteroarylhaving one or more heteroatoms selected from N, O, and S, —OR,—O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN, —C(O)N(R)₂,—N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂,—C(R)N(R)₂C(O)OR, and each n is independently selected from 1, 2, 3, 4,and 5.

In another embodiment, R₄ is unsubstituted C₁₋₄ alkyl, e.g.,unsubstituted methyl.

In certain embodiments, the disclosure provides a compound having theFormula (I), wherein R₄ is —(CH₂)_(n)Q or —(CH₂)_(n)CHQR, where Q is—N(R)₂, and n is selected from 3, 4, and 5.

In certain embodiments, the disclosure provides a compound having theFormula (I), wherein R₄ is selected from the group consisting of—(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, and —CQ(R)₂, where Q is —N(R)₂, andn is selected from 1, 2, 3, 4, and 5.

In certain embodiments, the disclosure provides a compound having theFormula (I), wherein R₂ and R₃ are independently selected from the groupconsisting of C₂₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, orR₂ and R₃, together with the atom to which they are attached, form aheterocycle or carbocycle, and R₄ is —(CH₂)_(n)Q or —(CH₂)_(n)CHQR,where Q is —N(R)₂, and n is selected from 3, 4, and 5.

In certain embodiments, R₂ and R₃ are independently selected from thegroup consisting of C₂₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and—R*OR″, or R₂ and R₃, together with the atom to which they are attached,form a heterocycle or carbocycle.

In some embodiments, R₁ is selected from the group consisting of C₅₋₂₀alkyl and C₅₋₂₀ alkenyl.

In other embodiments, R₁ is selected from the group consisting of—R*YR″, —YR″, and —R″M′R′.

In certain embodiments, R₁ is selected from —R*YR″ and —YR″. In someembodiments, Y is a cyclopropyl group. In some embodiments, R* is C₈alkyl or C₈ alkenyl. In certain embodiments, R″ is C₃₋₁₂ alkyl. Forexample, R″ can be C₃ alkyl. For example, R″ can be C₄₋₈ alkyl (e.g.,C₄, C₅, C₆, C₇, or C₈ alkyl).

In some embodiments, R₁ is C₅₋₂₀ alkyl. In some embodiments, R₁ is C₆alkyl. In some embodiments, R₁ is C₈ alkyl. In other embodiments, R₁ isC₉ alkyl. In certain embodiments, R₁ is C₁₄ alkyl. In other embodiments,R₁ is C₁₈ alkyl.

In some embodiments, R₁ is C₅₋₂₀ alkenyl. In certain embodiments, R₁ isC₁₈ alkenyl.

In some embodiments, R₁ is linoleyl.

In certain embodiments, R₁ is branched (e.g., decan-2-yl, undecan-3-yl,dodecan-4-yl, tridecan-5-yl, tetradecan-6-yl, 2-methylundecan-3-yl,2-methyldecan-2-yl, 3-methylundecan-3-yl, 4-methyldodecan-4-yl, orheptadeca-9-yl). In certain embodiments, R₁ is

In certain embodiments, R₁ is unsubstituted C₅₋₂₀ alkyl or C₅₋₂₀alkenyl. In certain embodiments, R′ is substituted C₅₋₂₀ alkyl or C₅₋₂₀alkenyl (e.g., substituted with a C₃₋₆ carbocycle such as1-cyclopropylnonyl).

In other embodiments, R₁ is —R″M′R′.

In some embodiments, R′ is selected from —R*YR″ and —YR″. In someembodiments, Y is C₃₋₈ cycloalkyl. In some embodiments, Y is C₆₋₁₀ aryl.In some embodiments, Y is a cyclopropyl group. In some embodiments, Y isa cyclohexyl group. In certain embodiments, R* is C₁ alkyl.

In some embodiments, R″ is selected from the group consisting of C₃₋₁₂alkyl and C₃₋₁₂ alkenyl. In some embodiments, R″ adjacent to Y is C₁alkyl. In some embodiments, R″ adjacent to Y is C₄₋₉ alkyl (e.g., C₄,C₅, C₆, C₇ or C₈ or C₉ alkyl).

In some embodiments, R′ is selected from C₄ alkyl and C₄ alkenyl. Incertain embodiments, R′ is selected from C₅ alkyl and C₅ alkenyl. Insome embodiments, R′ is selected from C₆ alkyl and C₆ alkenyl. In someembodiments, R′ is selected from C₇ alkyl and C₇ alkenyl. In someembodiments, R′ is selected from C₉ alkyl and C₉ alkenyl.

In other embodiments, R′ is selected from C₁₁ alkyl and C₁₁ alkenyl. Inother embodiments, R′ is selected from C₁₂ alkyl, C₁₂ alkenyl, C₁₃alkyl, C₁₃ alkenyl, C₁₄ alkyl, C₁₄ alkenyl, C₁₅ alkyl, C₁₅ alkenyl, C₁₆alkyl, C₁₆ alkenyl, C₁₇ alkyl, C₁₇ alkenyl, C₁₈ alkyl, and C₁₈ alkenyl.In certain embodiments, R′ is branched (e.g., decan-2-yl, undecan-3-yl,dodecan-4-yl, tridecan-5-yl, tetradecan-6-yl, 2-methylundecan-3-yl,2-methyldecan-2-yl, 3-methylundecan-3-yl, 4-methyldodecan-4-yl orheptadeca-9-yl). In certain embodiments, R′ is

In certain embodiments, R′ is unsubstituted C₁₋₁₈ alkyl. In certainembodiments, R′ is substituted C₁₋₈i alkyl (e.g., C₁₋₁₅ alkylsubstituted with a C₃₋₆ carbocycle such as 1-cyclopropylnonyl).

In some embodiments, R″ is selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl. In some embodiments, R″ is C₃ alkyl, C₄ alkyl,C₅ alkyl, C₆ alkyl, C₇ alkyl, or C₈ alkyl. In some embodiments, R″ is C₉alkyl, C₁₀ alkyl, C₁₁ alkyl, C₁₂ alkyl, C₁₃ alkyl, or C₁₄ alkyl.

In some embodiments, M′ is —C(O)O—. In some embodiments, M′ is —OC(O)—.

In other embodiments, M′ is an aryl group or heteroaryl group. Forexample, M′ can be selected from the group consisting of phenyl,oxazole, and thiazole.

In some embodiments, M is —C(O)O—. In some embodiments, M is —OC(O)—. Insome embodiments, M is —C(O)N(R′)—. In some embodiments, M is—P(O)(OR′)O—.

In other embodiments, M is an aryl group or heteroaryl group. Forexample, M can be selected from the group consisting of phenyl, oxazole,and thiazole.

In some embodiments, M is the same as M′. In other embodiments, M isdifferent from M′.

In some embodiments, each R₅ is H. In certain such embodiments, each R₆is also H.

In some embodiments, R₇ is H. In other embodiments, R₇ is C₁₋₃ alkyl(e.g., methyl, ethyl, propyl, or i-propyl).

In some embodiments, R₂ and R₃ are independently C₅₋₁₄ alkyl or C₅₋₁₄alkenyl.

In some embodiments, R₂ and R₃ are the same. In some embodiments, R₂ andR₃ are C₈ alkyl. In certain embodiments, R₂ and R₃ are C₂ alkyl. Inother embodiments, R₂ and R₃ are C₃ alkyl. In some embodiments, R₂ andR₃ are C₄ alkyl. In certain embodiments, R₂ and R₃ are C₅ alkyl. Inother embodiments, R₂ and R₃ are C₆ alkyl. In some embodiments, R₂ andR₃ are C₇ alkyl.

In other embodiments, R₂ and R₃ are different. In certain embodiments,R₂ is C₈ alkyl.

In some embodiments, R₃ is C₁₋₇ (e.g., C₁, C₂, C₃, C₄, C₅, C₆, or C₇alkyl) or C₉ alkyl.

In some embodiments, R₇ and R₃ are H.

In certain embodiments, R₂ is H.

In some embodiments, m is 5, 7, or 9.

In some embodiments, R₄ is selected from —(CH₂)_(n)Q and —(CH₂)_(n)CHQR.

In some embodiments, Q is selected from the group consisting of —OR,—OH, —O(CH₂)_(n)N(R)₂, —OC(O)R, —CX₃, —CN, —N(R)C(O)R, —N(H)C(O)R,—N(R)S(O)₂R, —N(H)S(O)₂R, —N(R)C(O)N(R)₂, —N(H)C(O)N(R)₂,—N(H)C(O)N(H)(R), —N(R)C(S)N(R)₂, —N(H)C(S)N(R)₂, —N(H)C(S)N(H)(R),—C(R)N(R)₂C(O)OR, a carbocycle, and a heterocycle.

In certain embodiments, Q is —OH.

In certain embodiments, Q is a substituted or unsubstituted 5- to10-membered heteroaryl, e.g., Q is an imidazole, a pyrimidine, a purine,2-amino-1,9-dihydro-6H-purin-6-one-9-yl (or guanin-9-yl), adenin-9-yl,cytosin-1-yl, or uracil-1-yl. In certain embodiments, Q is a substituted5- to 14-membered heterocycloalkyl, e.g., substituted with one or moresubstituents selected from oxo (═O), OH, amino, and C₁₋₃ alkyl. Forexample, Q is 4-methylpiperazinyl, 4-(4-methoxybenzyl)piperazinyl, orisoindolin-2-yl-1,3-dione.

In certain embodiments, Q is an unsubstituted or substituted C₆₋₁₀ aryl(such as phenyl) or C₃₋₆ cycloalkyl.

In some embodiments, n is 1. In other embodiments, n is 2. In furtherembodiments, n is 3. In certain other embodiments, n is 4. For example,R₄ can be —(CH₂)₂OH. For example, R₄ can be —(CH₂)₃OH. For example, R₄can be —(CH₂)₄OH. For example, R₄ can be benzyl. For example, R₄ can be4-methoxybenzyl.

In some embodiments, R₄ is a C₃₋₆ carbocycle. In some embodiments, R₄ isa C₃₋₆ cycloalkyl. For example, R₄ can be cyclohexyl optionallysubstituted with e.g., OH, halo, C₁₋₆ alkyl, etc. For example, R₄ can be2-hydroxycyclohexyl.

In some embodiments, R is H.

In some embodiments, R is unsubstituted C₁₋₃ alkyl or unsubstituted C₂₋₃alkenyl. For example, R₄ can be —CH₂CH(OH)CH₃ or —CH₂CH(OH)CH₂CH₃.

In some embodiments, R is substituted C₁₋₃ alkyl, e.g., CH₂OH. Forexample, R₄ can be —CH₂CH(OH)CH₂OH.

In some embodiments, R₂ and R₃, together with the atom to which they areattached, form a heterocycle or carbocycle. In some embodiments, R₂ andR₃, together with the atom to which they are attached, form a 5- to14-membered aromatic or non-aromatic heterocycle having one or moreheteroatoms selected from N, O, S, and P. In some embodiments, R₂ andR₃, together with the atom to which they are attached, form anoptionally substituted C₃₋₂₀ carbocycle (e.g., C₃₋₁₈ carbocycle, C₃₋₁₅carbocycle, C₃₋₁₂ carbocycle, or C₃₋₁₀ carbocycle), either aromatic ornon-aromatic. In some embodiments, R₂ and R₃, together with the atom towhich they are attached, form a C₃₋₆ carbocycle. In other embodiments,R₂ and R₃, together with the atom to which they are attached, form a C₆carbocycle, such as a cyclohexyl or phenyl group. In certainembodiments, the heterocycle or C₃₋₆ carbocycle is substituted with oneor more alkyl groups (e.g., at the same ring atom or at adjacent ornon-adjacent ring atoms). For example, R₂ and R₃, together with the atomto which they are attached, can form a cyclohexyl or phenyl groupbearing one or more C₅ alkyl substitutions. In certain embodiments, theheterocycle or C₃₋₆ carbocycle formed by R₂ and R₃, is substituted witha carbocycle groups. For example, R₂ and R₃, together with the atom towhich they are attached, can form a cyclohexyl or phenyl group that issubstituted with cyclohexyl. In some embodiments, R₂ and R₃, togetherwith the atom to which they are attached, form a C₇₋₁₅ carbocycle, suchas a cycloheptyl, cyclopentadecanyl, or naphthyl group.

In some embodiments, R₄ is selected from —(CH₂)_(n)Q and —(CH₂)_(n)CHQR.In some embodiments, Q is selected from the group consisting of —OR,—OH, —O(CH₂)_(n)N(R)₂, —OC(O)R, —CX₃, —CN, —N(R)C(O)R, —N(H)C(O)R,—N(R)S(O)₂R, —N(H)S(O)₂R, —N(R)C(O)N(R)₂, —N(H)C(O)N(R)₂,—N(H)C(O)N(H)(R), —N(R)C(S)N(R)₂, —N(H)C(S)N(R)₂, —N(H)C(S)N(H)(R), anda heterocycle. In other embodiments, Q is selected from the groupconsisting of an imidazole, a pyrimidine, and a purine.

In some embodiments, R₂ and R₃, together with the atom to which they areattached, form a heterocycle or carbocycle. In some embodiments, R₂ andR₃, together with the atom to which they are attached, form a C₃₋₆carbocycle, such as a phenyl group. In certain embodiments, theheterocycle or C₃₋₆ carbocycle is substituted with one or more alkylgroups (e.g., at the same ring atom or at adjacent or non-adjacent ringatoms). For example, R₂ and R₃, together with the atom to which they areattached, can form a phenyl group bearing one or more C₅ alkylsubstitutions.

In some embodiments, the pharmaceutical compositions of the presentdisclosure, the compound of formula (I) is selected from the groupconsisting of:

and salts and isomers thereof.

In other embodiments, the compound of Formula (I) is selected from thegroup consisting of Compound 1-Compound 147, or salt or stereoisomersthereof.

In some embodiments ionizable lipids including a central piperazinemoiety are provided. The lipids described herein may be advantageouslyused in lipid nanoparticle compositions for the delivery of therapeuticand/or prophylactic agents to mammalian cells or organs. For example,the lipids described herein have little or no immunogenicity. Forexample, the lipid compounds disclosed hereinhave a lower immunogenicityas compared to a reference lipid (e.g., MC3, KC2, or DLinDMA). Forexample, a formulation comprising a lipid disclosed herein and atherapeutic or prophylactic agent has an increased therapeutic index ascompared to a corresponding formulation which comprises a referencelipid (e.g., MC3, KC2, or DLinDMA) and the same therapeutic orprophylactic agent.

In some embodiments, the delivery agent comprises a lipid compoundhaving the formula (III)

or salts or stereoisomers thereof, wherein

ring A is

t is 1 or 2;

A₁ and A₂ are each independently selected from CH or N;

Z is CH₂ or absent wherein when Z is CH₂, the dashed lines (1) and (2)each represent a single bond; and when Z is absent, the dashed lines (1)and (2) are both absent;

R₁, R₂, R₃, R₄, and R₅ are independently selected from the groupconsisting of C₅₋₂₀ alkyl, C₅₋₂₀ alkenyl, —R″MR′, —R*YR″, —YR″, and—R*OR″;

each M is independently selected from the group consisting of —C(O)O—,—OC(O)—, —OC(O)O—, —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—,—SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O)₂—, an aryl group, and aheteroaryl group;

X¹, X², and X³ are independently selected from the group consisting of abond, —CH₂—, —(CH₂)₂—, —CHR—, —CHY—, —C(O)—, —C(O)O—, —OC(O)—,—C(O)—CH₂—, —CH₂—C(O)—, —C(O)O—CH₂—, —OC(O)—CH₂—, —CH₂—C(O)O—,—CH₂—OC(O)—, —CH(OH)—, —C(S)—, and —CH(SH—;

each Y is independently a C₃₋₆ carbocycle;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;

each R is independently selected from the group consisting of C₁₋₃ alkyland a C₃₋₆ carbocycle;

each R′ is independently selected from the group consisting of C₁₋₁₂alkyl, C₂₋₁₂ alkenyl, and H; and

each R″ is independently selected from the group consisting of C₃₋₁₂alkyl and C₃₋₁₂ alkenyl,

wherein when ring A is

then

i) at least one of X¹, X², and X³ is not —CH₂—; and/or

ii) at least one of R₁, R₂, R₃, R₄, and R₅ is —R″MR′.

In some embodiments, the compound is of any of formulae (IIIa1)-(IIIa6):

The compounds of Formula (III) or any of (IIIa1)-(IIIa6) include one ormore of the following features when applicable.

In some embodiments, ring A is

In some embodiments, ring A is

In some embodiments, ring A is

In some embodiments, ring A is

In some embodiments, ring A is

In some embodiments, ring A is

wherein ring, in which the N atom is connected with X².

In some embodiments, Z is CH₂.

In some embodiments, Z is absent.

In some embodiments, at least one of A₁ and A₂ is N.

In some embodiments, each of A₁ and A₂ is N.

In some embodiments, each of A₁ and A₂ is CH.

In some embodiments, A₁ is N and A₂ is CH.

In some embodiments, A₁ is CH and A₂ is N.

In some embodiments, at least one of X¹, X², and X³ is not —CH₂—. Forexample, in certain embodiments, X¹ is not —CH₂—. In some embodiments,at least one of X¹, X², and X³ is —C(O)—.

In some embodiments, X² is —C(O)—, —C(O)O—, —OC(O)—, —C(O)—CH₂—,—CH₂—C(O)—, —C(O)O—CH₂—, —OC(O)—CH₂—, —CH₂—C(O)O—, or —CH₂—OC(O)—.

In some embodiments, X³ is —C(O)—, —C(O)O—, —OC(O)—, —C(O)—CH₂—,—CH₂—C(O)—, —C(O)O—CH₂—, —OC(O)—CH₂—, —CH₂—C(O)O—, or —CH₂—OC(O)—. Inother embodiments, X³ is —CH₂—.

In some embodiments, X³ is a bond or —(CH₂)₂—.

In some embodiments, R₁ and R₂ are the same. In certain embodiments, R₁,R₂, and R₃ are the same. In some embodiments, R₄ and R₅ are the same. Incertain embodiments, R₁, R₂, R₃, R₄, and R₅ are the same.

In some embodiments, at least one of R₁, R₂, R₃, R₄, and R₅ is —R″MR′.In some embodiments, at most one of R₁, R₂, R₃, R₄, and R₅ is —R″MR′.For example, at least one of R₁, R₂, and R₃ may be —R″MR′, and/or atleast one of R₄ and R₅ is —R″MR′. In certain embodiments, at least one Mis —C(O)O—. In some embodiments, each M is —C(O)O—. In some embodiments,at least one M is —OC(O)—. In some embodiments, each M is —OC(O)—. Insome embodiments, at least one M is —OC(O)O—. In some embodiments, eachM is —OC(O)O—. In some embodiments, at least one R″ is C₃ alkyl. Incertain embodiments, each R″ is C₃ alkyl. In some embodiments, at leastone R″ is C₅ alkyl. In certain embodiments, each R″ is C₅ alkyl. In someembodiments, at least one R″ is C₆ alkyl. In certain embodiments, eachR″ is C₆ alkyl. In some embodiments, at least one R″ is C₇ alkyl. Incertain embodiments, each R″ is C₇ alkyl. In some embodiments, at leastone R′ is C₅ alkyl. In certain embodiments, each R′ is C₅ alkyl. Inother embodiments, at least one R′ is C₁ alkyl. In certain embodiments,each R′ is C₁ alkyl. In some embodiments, at least one R′ is C₂ alkyl.In certain embodiments, each R′ is C₂ alkyl.

In some embodiments, at least one of R₁, R₂, R₃, R₄, and R₅ is C₁₂alkyl. In certain embodiments, each of R₁, R₂, R₃, R₄, and R₅ are C₁₂alkyl.

In certain embodiments, the compound is selected from the groupconsisting of:

In some embodiments, the delivery agent comprises Compound 236.

In some embodiments, the delivery agent comprises a compound having theformula (IV)

or salts or stereoisomer thereof, wherein

A₁ and A₂ are each independently selected from CH or N and at least oneof A₁ and A₂ is N;

Z is CH₂ or absent wherein when Z is CH₂, the dashed lines (1) and (2)each represent a single bond; and when Z is absent, the dashed lines (1)and (2) are both absent;

R₁, R₂, R₃, R₄, and R₅ are independently selected from the groupconsisting of C₆₋₂₀ alkyl and C₆₋₂₀ alkenyl;

wherein when ring A is

then

i) R₁, R₂, R₃, R₄, and R₅ are the same, wherein R₁ is not C₁₂ alkyl, C₁₈alkyl, or C₁₈ alkenyl;

ii) only one of R₁, R₂, R₃, R₄, and R₅ is selected from C₆₋₂₀ alkenyl;

iii) at least one of R₁, R₂, R₃, R₄, and R₅ have a different number ofcarbon atoms than at least one other of R₁, R₂, R₃, R₄, and R₅;

iv) R₁, R₂, and R₃ are selected from C₆₋₂₀ alkenyl, and R₄ and R₅ areselected from C₆₋₂₀ alkyl; or

v) R₁, R₂, and R₃ are selected from C₆₋₂₀ alkyl, and R₄ and R₅ areselected from C₆₋₂₀ alkenyl.

In some embodiments, the compound is of formula (IVa):

The compounds of Formula (IV) or (IVa) include one or more of thefollowing features when applicable.

In some embodiments, Z is CH₂.

In some embodiments, Z is absent.

In some embodiments, at least one of A₁ and A₂ is N.

In some embodiments, each of A₁ and A₂ is N.

In some embodiments, each of A₁ and A₂ is CH.

In some embodiments, A₁ is N and A₂ is CH.

In some embodiments, A₁ is CH and A₂ is N.

In some embodiments, R₁, R₂, R₃, R₄, and R₅ are the same, and are notC₁₂ alkyl, C₁₈ alkyl, or C₁₈ alkenyl. In some embodiments, R₁, R₂, R₃,R₄, and R₅ are the same and are C₉ alkyl or C₁₄ alkyl.

In some embodiments, only one of R₁, R₂, R₃, R₄, and R₅ is selected fromC₆₋₂₀ alkenyl. In certain such embodiments, R₁, R₂, R₃, R₄, and R₅ havethe same number of carbon atoms. In some embodiments, R₄ is selectedfrom C₅₋₂₀ alkenyl. For example, R₄ may be C₁₂ alkenyl or C₁₈ alkenyl.

In some embodiments, at least one of R₁, R₂, R₃, R₄, and R₅ have adifferent number of carbon atoms than at least one other of R₁, R₂, R₃,R₄, and R₅.

In certain embodiments, R₁, R₂, and R₃ are selected from C₆₋₂₀ alkenyl,and R₄ and R₅ are selected from C₆₋₂₀ alkyl. In other embodiments, R₁,R₂, and R₃ are selected from C₆₋₂₀ alkyl, and R₄ and R₅ are selectedfrom C₆₋₂₀ alkenyl. In some embodiments, R₁, R₂, and R₃ have the samenumber of carbon atoms, and/or R₄ and R₅ have the same number of carbonatoms. For example, R₁, R₂, and R₃, or R₄ and R₅, may have 6, 8, 9, 12,14, or 18 carbon atoms. In some embodiments, R₁, R₂, and R₃, or R₄ andR₅, are C₁₈ alkenyl (e.g., linoleyl). In some embodiments, R₁, R₂, andR₃, or R₄ and R₅, are alkyl groups including 6, 8, 9, 12, or 14 carbonatoms.

In some embodiments, R₁ has a different number of carbon atoms than R₂,R₃, R₄, and R₅. In other embodiments, R₃ has a different number ofcarbon atoms than R₁, R₂, R₄, and R₅. In further embodiments, R₄ has adifferent number of carbon atoms than R₁, R₂, R₃, and R₅.

In some embodiments, the compound is selected from the group consistingof:

In other embodiments, the delivery agent comprises a compound having theformula (V)

or salts or stereoisomers thereof, in which

A₃ is CH or N;

A₄ is CH₂ or NH; and at least one of A₃ and A₄ is N or NH;

Z is CH₂ or absent wherein when Z is CH₂, the dashed lines (1) and (2)each represent a single bond; and when Z is absent, the dashed lines (1)and (2) are both absent;

R₁, R₂, and R₃ are independently selected from the group consisting ofC₅₋₂₀ alkyl, C₅₋₂₀ alkenyl, —R″MR′, —R*YR″, —YR″, and —R*OR″;

each M is independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, an aryl group, and a heteroaryl group;

X¹ and X² are independently selected from the group consisting of —CH₂—,—(CH₂)₂—, —CHR—, —CHY—, —C(O)—, —C(O)O—, —OC(O)—, —C(O)—CH₂—,—CH₂—C(O)—, —C(O)O—CH₂—, —OC(O)—CH₂—, —CH₂—C(O)O—, —CH₂—OC(O)—,—CH(OH)—, —C(S)—, and —CH(SH)—;

each Y is independently a C₃₋₆ carbocycle;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;

each R is independently selected from the group consisting of C₁₋₃ alkyland a C₃₋₆ carbocycle;

each R′ is independently selected from the group consisting of C₁₋₁₂alkyl, C₂₋₁₂ alkenyl, and H; and

each R″ is independently selected from the group consisting of C₃₋₁₂alkyl and C₃₋₁₂ alkenyl.

In some embodiments, the compound is of formula (Va):

The compounds of Formula (V) or (Va) include one or more of thefollowing features when applicable.

In some embodiments, Z is CH₂.

In some embodiments, Z is absent.

In some embodiments, at least one of A₃ and A₄ is N or NH.

In some embodiments, A₃ is N and A₄ is NH.

In some embodiments, A₃ is N and A₄ is CH₂.

In some embodiments, A₃ is CH and A₄ is NH.

In some embodiments, at least one of X¹ and X² is not —CH₂—. Forexample, in certain embodiments, X¹ is not —CH₂—. In some embodiments,at least one of X¹ and X² is —C(O)—.

In some embodiments, X² is —C(O)—, —C(O)O—, —OC(O)—, —C(O)—CH₂—,—CH₂—C(O)—, —C(O)O—CH₂—, —OC(O)—CH₂—, —CH₂—C(O)O—, or —CH₂—OC(O)—.

In some embodiments, R₁, R₂, and R₃ are independently selected from thegroup consisting of C₅₋₂₀ alkyl and C₅₋₂₀ alkenyl. In some embodiments,R₁, R₂, and R₃ are the same. In certain embodiments, R₁, R₂, and R₃ areC₆, C₉, C₁₂, or C₁₄ alkyl. In other embodiments, R₁, R₂, and R₃ are C₁₈alkenyl. For example, R₁, R₂, and R₃ may be linoleyl.

In some embodiments, the compound is selected from the group consistingof:

In other embodiments, the delivery agent comprises a compound having theformula (VI):

or salts or stereoisomers thereof, in which

A₆ and A₇ are each independently selected from CH or N, wherein at leastone of A₆ and A₇ is N;

Z is CH₂ or absent wherein when Z is CH₂, the dashed lines (1) and (2)each represent a single bond; and when Z is absent, the dashed lines (1)and (2) are both absent;

X⁴ and X⁵ are independently selected from the group consisting of —CH₂—,—CH₂)₂—, —CHR—, —CHY—, —C(O)—, —C(O)O—, —OC(O)—, —C(O)—CH₂—, —CH₂—C(O)—,—C(O)O—CH₂—, —OC(O)—CH₂—, —CH₂—C(O)O—, —CH₂—OC(O)—, —CH(OH)—, —C(S)—,and —CH(SH)—;

R₁, R₂, R₃, R₄, and R₅ each are independently selected from the groupconsisting of C₅₋₂₀ alkyl, C₅₋₂₀ alkenyl, —R″MR′, —R*YR″, —YR″, and—R*OR″;

each M is independently selected from the group consisting of —C(O)O—,—OC(O)—, —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—,—CH(OH)—, —P(O)(OR′)O—, —S(O)₂— an aryl group, and a heteroaryl group;

each Y is independently a C₃₋₆ carbocycle;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;

each R is independently selected from the group consisting of C₁₋₃ alkyland a C₃₋₆ carbocycle;

each R′ is independently selected from the group consisting of C₁₋₁₂alkyl, C₂₋₁₂ alkenyl, and H; and

each R″ is independently selected from the group consisting of C₃₋₁₂alkyl and C₃₋₁₂ alkenyl.

In some embodiments, R₁, R₂, R₃, R₄, and R₅ each are independentlyselected from the group consisting of C₆₋₂₀ alkyl and C₆₋₂₀ alkenyl.

In some embodiments, R₁ and R₂ are the same. In certain embodiments, R₁,R₂, and R₃ are the same. In some embodiments, R₄ and R₅ are the same. Incertain embodiments, R₁, R₂, R₃, R₄, and R₅ are the same.

In some embodiments, at least one of R₁, R₂, R₃, R₄, and R₅ is C₉₋₁₂alkyl. In certain embodiments, each of R₁, R₂, R₃, R₄, and R₅independently is C₉, C₁₂ or C₁₄ alkyl. In certain embodiments, each ofR₁, R₂, R₃, R₄, and R₅ is C₉ alkyl.

In some embodiments, A₆ is N and A₇ is N. In some embodiments, A₆ is CHand A₇ is N.

In some embodiments, X⁴ is —CH₂— and X⁵ is —C(O)—. In some embodiments,X⁴ and X⁵ are —C(O)—.

In some embodiments, when A₆ is N and A₇ is N, at least one of X⁴ and X⁵is not —CH₂—, e.g., at least one of X⁴ and X⁵ is —C(O)—. In someembodiments, when A₆ is N and A₇ is N, at least one of R₁, R₂, R₃, R₄,and R₅ is —R″MR′.

In some embodiments, at least one of R₁, R₂, R₃, R₄, and R₅ is not—R″MR′.

In some embodiments, the compound is

In other embodiments, the delivery agent comprises a compound having theformula:

Amine moieties of the lipid compounds disclosed herein can be protonatedunder certain conditions. For example, the central amine moiety of alipid according to formula (I) is typically protonated (i.e., positivelycharged) at a pH below the pKa of the amino moiety and is substantiallynot charged at a pH above the pKa. Such lipids can be referred toionizable amino lipids.

In one specific embodiment, the ionizable amino lipid is Compound 18. Inanother embodiment, the ionizable amino lipid is Compound 236.

In some embodiments, the amount the ionizable amino lipid, e.g.,compound of formula (I) ranges from about 1 mol % to 99 mol % in thelipid composition.

In one embodiment, the amount of the ionizable amino lipid, e.g.,compound of formula (I) is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99mol % in the lipid composition.

In one embodiment, the amount of the ionizable amino lipid, e.g., thecompound of formula (I) ranges from about 30 mol % to about 70 mol %,from about 35 mol % to about 65 mol %, from about 40 mol % to about 60mol %, and from about 45 mol % to about 55 mol % in the lipidcomposition.

In one specific embodiment, the amount of the ionizable amino lipid,e.g., compound of formula (I) is about 50 mol % in the lipidcomposition.

In addition to the ionizable amino lipid disclosed herein, e.g.,compound of formula (I), the lipid composition of the pharmaceuticalcompositions disclosed herein can comprise additional components such asphospholipids, structural lipids, PEG-lipids, and any combinationthereof.

b. Phospholipids

The lipid composition of the pharmaceutical composition disclosed hereincan comprise one or more phospholipids, for example, one or moresaturated or (poly)unsaturated phospholipids or a combination thereof.In general, phospholipids comprise a phospholipid moiety and one or morefatty acid moieties.

A phospholipid moiety can be selected, for example, from thenon-limiting group consisting of phosphatidyl choline, phosphatidylethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidicacid, 2-lysophosphatidyl choline, and a sphingomyelin.

A fatty acid moiety can be selected, for example, from the non-limitinggroup consisting of lauric acid, myristic acid, myristoleic acid,palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleicacid, alpha-linolenic acid, erucic acid, phytanoic acid, arachidic acid,arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoicacid, and docosahexaenoic acid.

Particular phospholipids can facilitate fusion to a membrane. Forexample, a cationic phospholipid can interact with one or morenegatively charged phospholipids of a membrane (e.g., a cellular orintracellular membrane). Fusion of a phospholipid to a membrane canallow one or more elements (e.g., a therapeutic agent) of alipid-containing composition (e.g., LNPs) to pass through the membranepermitting, e.g., delivery of the one or more elements to a targettissue.

Non-natural phospholipid species including natural species withmodifications and substitutions including branching, oxidation,cyclization, and alkynes are also contemplated. For example, aphospholipid can be functionalized with or cross-linked to one or morealkynes (e.g., an alkenyl group in which one or more double bonds isreplaced with a triple bond). Under appropriate reaction conditions, analkyne group can undergo a copper-catalyzed cycloaddition upon exposureto an azide. Such reactions can be useful in functionalizing a lipidbilayer of a nanoparticle composition to facilitate membrane permeationor cellular recognition or in conjugating a nanoparticle composition toa useful component such as a targeting or imaging moiety (e.g., a dye).

Phospholipids include, but are not limited to, glycerophospholipids suchas phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines,phosphatidylinositols, phosphatidy glycerols, and phosphatidic acids.Phospholipids also include phosphosphingolipid, such as sphingomyelin.

Examples of phospholipids include, but are not limited to, thefollowing:

In certain embodiments, a phospholipid useful or potentially useful inthe present invention is an analog or variant of DSPC(1,2-dioctadecanoyl-sn-glycero-3-phosphocholine). In certainembodiments, a phospholipid useful or potentially useful in the presentinvention is a compound of Formula (IX):

(or a salt thereof, wherein:

each R¹ is independently optionally substituted alkyl; or optionally twoR¹ are joined together with the intervening atoms to form optionallysubstituted monocyclic carbocyclyl or optionally substituted monocyclicheterocyclyl; or optionally three R¹ are joined together with theintervening atoms to form optionally substituted bicyclic carbocyclyl oroptionally substitute bicyclic heterocyclyl;

n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

A is of the formula:

each instance of L² is independently a bond or optionally substitutedC₁₋₆ alkylene, wherein one methylene unit of the optionally substitutedC₁₋₆ alkylene is optionally replaced with —O—, —N(R^(N))—, —S—, —C(O)—,—C(O)N(R^(N))—, —NR^(N)C(O)—, —C(O)O—, —OC(O)—, —OC(O)O—,—OC(O)N(R^(N))—, —NR^(N)C(O)O—, or —NR^(N)C(O)N(R^(N))—;

each instance of R² is independently optionally substituted C₁₋₃₀ alkyl,optionally substituted C₁₋₃₀ alkenyl, or optionally substituted C₁₋₃₀alkynyl; optionally wherein one or more methylene units of R² areindependently replaced with optionally substituted carbocyclylene,optionally substituted heterocyclylene, optionally substituted arylene,optionally substituted heteroarylene, —N(R^(N))—, —O—, —S—, —C(O)—,—C(O)N(R^(N))—, —NR^(N)C(O)—, —NR^(N)C(O)N(R^(N))—, —C(O)O—, —OC(O)—,—OC(O)O—, —OC(O)N(R^(N))—, —NR^(N)C(O)O—, —C(O)S—, —SC(O)—,—C(═NR^(N))—, —C(═NR^(N))N(R^(N))—, —NR^(N)C(═NR^(N))—,—NR^(N)C(═NR^(N))N(R^(N))—, —C(S)—, —C(S)N(R^(N))—, —NR^(N)C(S)—,—NR^(N)C(S)N(R^(N))—, —S(O)—, —OS(O)—, —S(O)O—, —OS(O)O—, —OS(O)₂—,—S(O)₂O—, —OS(O)₂O—, —N(R^(N))S(O)—, —S(O)N(R^(N))—,—N(R^(N))S(O)N(R^(N))—, —OS(O)N(R^(N))—, —N(R^(N))S(O)O—, —S(O)₂—,—N(R^(N))S(O)₂₋, —S(O)₂N(R^(N))—, —N(R^(N))S(O)₂N(R^(N))—,—OS(O)₂N(R^(N))—, or —N(R^(N))S(O)₂O—;

each instance of R^(N) is independently hydrogen, optionally substitutedalkyl, or a nitrogen protecting group;

Ring B is optionally substituted carbocyclyl, optionally substitutedheterocyclyl, optionally substituted aryl, or optionally substitutedheteroaryl; and

p is 1 or 2;

provided that the compound is not of the formula:

wherein each instance of R² is independently unsubstituted alkyl,unsubstituted alkenyl, or unsubstituted alkynyl.

i) Phospholipid Head Modifications

In certain embodiments, a phospholipid useful or potentially useful inthe present invention comprises a modified phospholipid head (e.g., amodified choline group). In certain embodiments, a phospholipid with amodified head is DSPC, or analog thereof, with a modified quaternaryamine. For example, in embodiments of Formula (IX), at least one of R¹is not methyl. In certain embodiments, at least one of R¹ is nothydrogen or methyl. In certain embodiments, the compound of Formula (IX)is of one of the following formulae:

or a salt thereof, wherein:

each t is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

each u is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and

each v is independently 1, 2, or 3.

In certain embodiments, the compound of Formula (IX) is of one of thefollowing formulae:

or a salt thereof.

In certain embodiments, a compound of Formula (IX) is one of thefollowing:

or a salt thereof.

In certain embodiments, a compound of Formula (IX) is of Formula (IX-a):

or a salt thereof.

In certain embodiments, phospholipids useful or potentially useful inthe present invention comprise a modified core. In certain embodiments,a phospholipid with a modified core described herein is DSPC, or analogthereof, with a modified core structure. For example, in certainembodiments of Formula (IX-a), group A is not of the following formula:

In certain embodiments, the compound of Formula (IX-a) is of one of thefollowing formulae:

or a salt thereof.

In certain embodiments, a compound of Formula (IX) is one of thefollowing:

or salts thereof.

In certain embodiments, a phospholipid useful or potentially useful inthe present invention comprises a cyclic moiety in place of theglyceride moiety. In certain embodiments, a phospholipid useful in thepresent invention is DSPC(1,2-dioctadecanoyl-sn-glycero-3-phosphocholine), or analog thereof,with a cyclic moiety in place of the glyceride moiety. In certainembodiments, the compound of Formula (IX) is of Formula (IX-b):

or a salt thereof.

In certain embodiments, the compound of Formula (IX-b) is of Formula(IX-b-1):

or a salt thereof, wherein:

w is 0, 1, 2, or 3.

In certain embodiments, the compound of Formula (IX-b) is of Formula(IX-b-2):

or a salt thereof.

In certain embodiments, the compound of Formula (IX-b) is of Formula(IX-b-3):

or a salt thereof.

In certain embodiments, the compound of Formula (IX-b) is of Formula(IX-b-4):

or a salt thereof.

In certain embodiments, the compound of Formula (IX-b) is one of thefollowing:

-   -   or salts thereof.

(ii) Phospholipid Tail Modifications

In certain embodiments, a phospholipid useful or potentially useful inthe present invention comprises a modified tail. In certain embodiments,a phospholipid useful or potentially useful in the present invention isDSPC (1,2-dioctadecanoyl-sn-glycero-3-phosphocholine), or analogthereof, with a modified tail. As described herein, a “modified tail”may be a tail with shorter or longer aliphatic chains, aliphatic chainswith branching introduced, aliphatic chains with substituentsintroduced, aliphatic chains wherein one or more methylenes are replacedby cyclic or heteroatom groups, or any combination thereof. For example,in certain embodiments, the compound of (IX) is of Formula (IX-a), or asalt thereof, wherein at least one instance of R² is each instance of R²is optionally substituted C₁₋₃₀ alkyl, wherein one or more methyleneunits of R² are independently replaced with optionally substitutedcarbocyclylene, optionally substituted heterocyclylene, optionallysubstituted arylene, optionally substituted heteroarylene, —N(R^(N))—,—O—, —S—, —C(O)—, —C(O)N(R^(N))—, —NR^(N)C(O)—, —NR^(N)C(O)N(R^(N))—,—C(O)O—, —OC(O)—, —OC(O)O—, —OC(O)N(R^(N))—, —NR^(N)C(O)O—, —C(O)S—,—SC(O)—, —C(═NR^(N))—, —C(═NR^(N))N(R^(N))—, —NR^(N)C(═NR^(N))—,—NR^(N)C(═NR^(N))N(R^(N))—, —C(S)—, —C(S)N(R^(N))—, —NR^(N)C(S)—,—NR^(N)C(S)N(R^(N))—, —S(O)—, —OS(O)—, —S(O)O—, —OS(O)O—, —OS(O)₂—,—S(O)₂O—, —OS(O)₂O—, —N(R^(N))S(O)—, —S(O)N(R^(N))—,—N(R^(N))S(O)N(R^(N))—, —OS(O)N(R^(N))—, —N(R^(N))S(O)O—, —S(O)₂—,—N(R^(N))S(O)₂—, —S(O)₂N(R^(N))—, —N(R^(N))S(O)₂N(R^(N))—,—OS(O)₂N(R^(N))—, or —N(R^(N))S(O)₂O—.

In certain embodiments, the compound of Formula (IX) is of Formula(IX-c):

or a salt thereof, wherein:

each x is independently an integer between 0-30, inclusive; and

each instance is G is independently selected from the group consistingof optionally substituted carbocyclylene, optionally substitutedheterocyclylene, optionally substituted arylene, optionally substitutedheteroarylene, —N(R^(N))—, —O—, —S—, —C(O)—, —C(O)N(R^(N))—,—NR^(N)C(O)—, —NR^(N)C(O)N(R^(N))—, —C(O)O—, —OC(O)—, —OC(O)O—,—OC(O)N(R^(N))—, —NR^(N)C(O)O—, —C(O)S—, —SC(O)—, —C(═NR^(N))—,—C(═NR^(N))N(R^(N))—, —NR^(N)C(═NR^(N))—, —NR^(N)C(═NR^(N))N(R^(N))—,—C(S)—, —C(S)N(R^(N))—, —NR^(N)C(S)—, —NR^(N)C(S)N(R^(N))—, —S(O)—,—OS(O)—, —S(O)O—, —OS(O)O—, —OS(O)₂—, —S(O)₂O—, —OS(O)₂O—,—N(R^(N))S(O)—, —S(O)N(R^(N))—, —N(R^(N))S(O)N(R^(N))—, —OS(O)N(R^(N))—,—N(R^(N))S(O)O—, —S(O)₂—, —N(R^(N))S(O)₂—, —S(O)₂N(R^(N))—,—N(R^(N))S(O)₂N(R^(N))—, —OS(O)₂N(R^(N))—, or —N(R^(N))S(O)₂O—. Eachpossibility represents a separate embodiment of the present invention.

In certain embodiments, the compound of Formula (IX-c) is of Formula(IX-c-1):

or salt thereof, wherein:

each instance of v is independently 1, 2, or 3.

In certain embodiments, the compound of Formula (IX-c) is of Formula(IX-c-2):

or a salt thereof.

In certain embodiments, the compound of Formula (IX-c) is of thefollowing formula:

or a salt thereof.

In certain embodiments, the compound of Formula (IX-c) is the following:

or a salt thereof.

In certain embodiments, the compound of Formula (IX-c) is of Formula(IX-c-3):

or a salt thereof.

In certain embodiments, the compound of Formula (IX-c) is of thefollowing formulae:

or a salt thereof.

In certain embodiments, the compound of Formula (IX-c) is the following:

or a salt thereof.

In certain embodiments, a phospholipid useful or potentially useful inthe present invention comprises a modified phosphocholine moiety,wherein the alkyl chain linking the quaternary amine to the phosphorylgroup is not ethylene (e.g., n is not 2). Therefore, in certainembodiments, a phospholipid useful or potentially useful in the presentinvention is a compound of Formula (IX), wherein n is 1, 3, 4, 5, 6, 7,8, 9, or 10. For example, in certain embodiments, a compound of Formula(IX) is of one of the following formulae:

or a salt thereof.

In certain embodiments, a compound of Formula (IX) is one of thefollowing:

or salts thereof.

c. Alternative Lipids

In certain embodiments, an alternative lipid is used in place of aphospholipid of the invention. Non-limiting examples of such alternativelipids include the following:

d. Structural Lipids

The lipid composition of a pharmaceutical composition disclosed hereincan comprise one or more structural lipids. As used herein, the term“structural lipid” refers to sterols and also to lipids containingsterol moieties.

Incorporation of structural lipids in the lipid nanoparticle may helpmitigate aggregation of other lipids in the particle. Structural lipidscan be selected from the group including but not limited to,cholesterol, fecosterol, sitosterol, ergosterol, campesterol,stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid,alpha-tocopherol, hopanoids, phytosterols, steroids, and mixturesthereof. In some embodiments, the structural lipid is a sterol. Asdefined herein, “sterols” are a subgroup of steroids consisting ofsteroid alcohols. In certain embodiments, the structural lipid is asteroid. In certain embodiments, the structural lipid is cholesterol. Incertain embodiments, the structural lipid is an analog of cholesterol.In certain embodiments, the structural lipid is alpha-tocopherol.Examples of structural lipids include, but are not limited to, thefollowing:

In one embodiment, the amount of the structural lipid (e.g., an sterolsuch as cholesterol) in the lipid composition of a pharmaceuticalcomposition disclosed herein ranges from about 20 mol % to about 60 mol%, from about 25 mol % to about 55 mol %, from about 30 mol % to about50 mol %, or from about 35 mol % to about 45 mol %.

In one embodiment, the amount of the structural lipid (e.g., an sterolsuch as cholesterol) in the lipid composition disclosed herein rangesfrom about 25 mol % to about 30 mol %, from about 30 mol % to about 35mol %, or from about 35 mol % to about 40 mol %.

In one embodiment, the amount of the structural lipid (e.g., a sterolsuch as cholesterol) in the lipid composition disclosed herein is about24 mol %, about 29 mol %, about 34 mol %, or about 39 mol %.

In some embodiments, the amount of the structural lipid (e.g., an sterolsuch as cholesterol) in the lipid composition disclosed herein is atleast about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, or 60 mol %.

e. Polyethylene Glycol (PEG)-Lipids

The lipid composition of a pharmaceutical composition disclosed hereincan comprise one or more a polyethylene glycol (PEG) lipid.

As used herein, the term “PEG-lipid” refers to polyethylene glycol(PEG)-modified lipids. Non-limiting examples of PEG-lipids includePEG-modified phosphatidylethanolamine and phosphatidic acid,PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modifieddialkylamines and PEG-modified 1,2-diacyloxypropan-3-amines. Such lipidsare also referred to as PEGylated lipids. For example, a PEG lipid canbe PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPElipid.

In some embodiments, the PEG-lipid includes, but not limited to1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG),1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)] (PEG-DSPE), PEG-disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl,PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG-DAG),PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), orPEG-1,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA).

In one embodiment, the PEG-lipid is selected from the group consistingof a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidicacid, a PEG-modified ceramide, a PEG-modified dialkylamine, aPEG-modified diacylglycerol, a PEG-modified dialkylglycerol, andmixtures thereof.

In some embodiments, the lipid moiety of the PEG-lipids includes thosehaving lengths of from about C₁₄ to about C₂₂, preferably from about C₁₄to about C₁₆. In some embodiments, a PEG moiety, for example anmPEG-NH₂, has a size of about 1000, 2000, 5000, 10,000, 15,000 or 20,000daltons. In one embodiment, the PEG-lipid is PEG_(2k)-DMG.

In one embodiment, the lipid nanoparticles described herein can comprisea PEG lipid which is a non-diffusible PEG. Non-limiting examples ofnon-diffusible PEGs include PEG-DSG and PEG-DSPE.

PEG-lipids are known in the art, such as those described in U.S. Pat.No. 8,158,601 and International Publ. No. WO 2015/130584 A2, which areincorporated herein by reference in their entirety.

In general, some of the other lipid components (e.g., PEG lipids) ofvarious formulae, described herein may be synthesized as describedInternational Patent Application No. PCT/US2016/000129, filed Dec. 10,2016, entitled “Compositions and Methods for Delivery of TherapeuticAgents,” which is incorporated herein by reference in its entirety.

The lipid component of a lipid nanoparticle composition may include oneor more molecules comprising polyethylene glycol, such as PEG orPEG-modified lipids. Such species may be alternately referred to asPEGylated lipids. A PEG lipid is a lipid modified with polyethyleneglycol. A PEG lipid may be selected from the non-limiting groupincluding PEG-modified phosphatidylethanolamines, PEG-modifiedphosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines,PEG-modified diacylglycerols, PEG-modified dialkylglycerols, andmixtures thereof. For example, a PEG lipid may be PEG-c-DOMG, PEG-DMG,PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.

In some embodiments the PEG-modified lipids are a modified form of PEGDMG. PEG-DMG has the following structure:

In one embodiment, PEG lipids useful in the present invention can bePEGylated lipids described in International Publication No.WO2012099755, the contents of which is incorporated herein by referencein its entirety. Any of these exemplary PEG lipids described herein maybe modified to comprise a hydroxyl group on the PEG chain. In certainembodiments, the PEG lipid is a PEG-OH lipid. As generally definedherein, a “PEG-OH lipid” (also referred to herein as “hydroxy-PEGylatedlipid”) is a PEGylated lipid having one or more hydroxyl (—OH) groups onthe lipid. In certain embodiments, the PEG-OH lipid includes one or morehydroxyl groups on the PEG chain. In certain embodiments, a PEG-OH orhydroxy-PEGylated lipid comprises an —OH group at the terminus of thePEG chain. Each possibility represents a separate embodiment of thepresent invention.

In certain embodiments, a PEG lipid useful in the present invention is acompound of Formula (VII). Provided herein are compounds of Formula(VII):

or salts thereof, wherein:

R³ is —OR^(O);

R^(O) is hydrogen, optionally substituted alkyl, or an oxygen protectinggroup;

r is an integer between 1 and 100, inclusive;

L¹ is optionally substituted C₁₋₁₀ alkylene, wherein at least onemethylene of the optionally substituted C₁₋₁₀ alkylene is independentlyreplaced with optionally substituted carbocyclylene, optionallysubstituted heterocyclylene, optionally substituted arylene, optionallysubstituted heteroarylene, O, N(R^(N)), S, C(O), C(O)N(R^(N)),NR^(N)C(O), C(O)O, —OC(O), OC(O)O, OC(O)N(R^(N)), NR^(N)C(O)O, orNR^(N)C(O)N(R^(N));

D is a moiety obtained by click chemistry or a moiety cleavable underphysiological conditions;

m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

A is of the formula:

each instance of L² is independently a bond or optionally substitutedC₁₋₆ alkylene, wherein one methylene unit of the optionally substitutedC₁₋₆ alkylene is optionally replaced with O, N(R^(N)), S, C(O),C(O)N(R^(N)), NR^(N)C(O), C(O)O, OC(O), OC(O)O, OC(O)N(R^(N)),—NR^(N)C(O)O, or NR^(N)C(O)N(R^(N));

each instance of R² is independently optionally substituted C₁₋₃₀ alkyl,optionally substituted C₁₋₃₀ alkenyl, or optionally substituted C₁₋₃₀alkynyl; optionally wherein one or more methylene units of R² areindependently replaced with optionally substituted carbocyclylene,optionally substituted heterocyclylene, optionally substituted arylene,optionally substituted heteroarylene, N(R^(N)), O, S, C(O),C(O)N(R^(N)), NR^(N)C(O), —NR^(N)C(O)N(R^(N)), C(O)O, OC(O), OC(O)O,OC(O)N(R^(N)), NR^(N)C(O)O, C(O)S, SC(O), —C(═NR^(N)),C(═NR^(N))N(R^(N)), NR^(N)C(═NR^(N)), NR^(N)C(═NR^(N))N(R^(N)), C(S),C(S)N(R^(N)), NR^(N)C(S), NR^(N)C(S)N(R^(N)), S(O), OS(O), S(O)O,OS(O)O, OS(O)₂, S(O)₂₀, OS(O)₂₀, N(R^(N))S(O), —S(O)N(R^(N)),N(R^(N))S(O)N(R^(N)), OS(O)N(R^(N)), N(R^(N))S(O)O, S(O)₂,N(R^(N))S(O)₂, S(O)₂N(R^(N)), N(R^(N))S(O)₂N(R^(N)), OS(O)₂N(R^(N)), orN(R^(N))S(O)₂O;

each instance of R^(N) is independently hydrogen, optionally substitutedalkyl, or a nitrogen protecting group;

Ring B is optionally substituted carbocyclyl, optionally substitutedheterocyclyl, optionally substituted aryl, or optionally substitutedheteroaryl; and

p is 1 or 2.

In certain embodiments, the compound of Formula (VII) is a PEG-OH lipid(i.e., R³ is —OR^(O), and R^(O) is hydrogen). In certain embodiments,the compound of Formula (VII) is of Formula (VII-OH):

or a salt thereof.

In certain embodiments, D is a moiety obtained by click chemistry (e.g.,triazole). In certain embodiments, the compound of Formula (VII) is ofFormula (VII-a-1) or (VII-a-2):

or a salt thereof.

In certain embodiments, the compound of Formula (VII) is of one of thefollowing formulae:

or a salt thereof, wherein

s is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In certain embodiments, the compound of Formula (VII) is of one of thefollowing formulae:

or a salt thereof.

In certain embodiments, a compound of Formula (VII) is of one of thefollowing formulae:

or a salt thereof.

In certain embodiments, a compound of Formula (VII) is of one of thefollowing formulae:

or a salt thereof.

In certain embodiments, D is a moiety cleavable under physiologicalconditions (e.g., ester, amide, carbonate, carbamate, urea). In certainembodiments, a compound of Formula (VII) is of Formula (VII-b-1) or(VII-b-2):

or a salt thereof.

In certain embodiments, a compound of Formula (VII) is of Formula(VII-b-1-OH) or (VII-b-2-OH):

or a salt thereof.

In certain embodiments, the compound of Formula (VII) is of one of thefollowing formulae:

or a salt thereof.

In certain embodiments, a compound of Formula (VII) is of one of thefollowing formulae:

or a salt thereof.

In certain embodiments, a compound of Formula (VII) is of one of thefollowing formulae:

or a salt thereof.

In certain embodiments, a compound of Formula (VII) is of one of thefollowing formulae:

or salts thereof.

In certain embodiments, a PEG lipid useful in the present invention is aPEGylated fatty acid. In certain embodiments, a PEG lipid useful in thepresent invention is a compound of Formula (VIII). Provided herein arecompounds of Formula (VIII):

or a salts thereof, wherein:

R³ is —OR^(O);

R^(O) is hydrogen, optionally substituted alkyl or an oxygen protectinggroup;

r is an integer between 1 and 100, inclusive;

R⁵ is optionally substituted C₁₀₋₄₀ alkyl, optionally substituted C₁₀₋₄₀alkenyl, or optionally substituted C₁₀₋₄₀ alkynyl; and optionally one ormore methylene groups of R⁵ are replaced with optionally substitutedcarbocyclylene, optionally substituted heterocyclylene, optionallysubstituted arylene, optionally substituted heteroarylene, N(R^(N)), O,S, C(O), —C(O)N(R^(N)), NR^(N)C(O), NR^(N)C(O)N(R^(N)), C(O)O, OC(O),OC(O)O, OC(O)N(R^(N)), —NR^(N)C(O)O, C(O)S, SC(O), C(═NR^(N)),C(═NR^(N))N(R^(N)), NR^(N)C(═NR^(N)), NR^(N)C(═NR^(N))N(R^(N)), —C(S),C(S)N(R^(N)), NR^(N)C(S), NR^(N)C(S)N(R^(N)), S(O), OS(O), S(O)O,OS(O)O, OS(O)₂, —S(O)₂O, OS(O)₂O, N(R^(N))S(O), S(O)N(R^(N)),N(R^(N))S(O)N(R^(N)), OS(O)N(R^(N)), N(R^(N))S(O)O, —S(O)₂,N(R^(N))S(O)₂, S(O)₂N(R^(N)), N(R^(N))S(O)₂N(R^(N)), OS(O)₂N(R^(N)), orN(R^(N))S(O)₂O; and

each instance of R^(N) is independently hydrogen, optionally substitutedalkyl, or a nitrogen protecting group.

In certain embodiments, the compound of Formula (VIII) is of Formula(VIII-OH):

or a salt thereof. In some embodiments, r is 45.

In certain embodiments, a compound of Formula (VIII) is of one of thefollowing formulae:

or a salt thereof. In some embodiments, r is 45.

In yet other embodiments the compound of Formula (VIII) is:

or a salt thereof.

In one embodiment, the compound of Formula (VIII) is

In one embodiment, the amount of PEG-lipid in the lipid composition of apharmaceutical composition disclosed herein ranges from about 0.1 mol %to about 5 mol %, from about 0.5 mol % to about 5 mol %, from about 1mol % to about 5 mol %, from about 1.5 mol % to about 5 mol %, fromabout 2 mol % to about 5 mol % mol %, from about 0.1 mol % to about 4mol %, from about 0.5 mol % to about 4 mol %, from about 1 mol % toabout 4 mol %, from about 1.5 mol % to about 4 mol %, from about 2 mol %to about 4 mol %, from about 0.1 mol % to about 3 mol %, from about 0.5mol % to about 3 mol %, from about 1 mol % to about 3 mol %, from about1.5 mol % to about 3 mol %, from about 2 mol % to about 3 mol %, fromabout 0.1 mol % to about 2 mol %, from about 0.5 mol % to about 2 mol %,from about 1 mol % to about 2 mol %, from about 1.5 mol % to about 2 mol%, from about 0.1 mol % to about 1.5 mol %, from about 0.5 mol % toabout 1.5 mol %, or from about 1 mol % to about 1.5 mol %.

In one embodiment, the amount of PEG-lipid in the lipid compositiondisclosed herein is about 2 mol %. In one embodiment, the amount ofPEG-lipid in the lipid composition disclosed herein is about 1.5 mol %.

In one embodiment, the amount of PEG-lipid in the lipid compositiondisclosed herein is at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2,2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7,3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5 mol %.

In some aspects, the lipid composition of the pharmaceuticalcompositions disclosed herein does not comprise a PEG-lipid.

f. Other Ionizable Amino Lipids

The lipid composition of the pharmaceutical composition disclosed hereincan comprise one or more ionizable amino lipids in addition to orinstead of a lipid according to Formula (I), (II), (III), (IV), (V), or(VI).

Ionizable lipids can be selected from the non-limiting group consistingof 3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10),N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine(KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25),1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA),2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA),heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate(DLin-MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane(DLin-KC2-DMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA),(13Z,165Z)—N,N-dimethyl-3-nonydocosa-13-16-dien-1-amine (L608),2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine(Octyl-CLinDMA),(2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine(Octyl-CLinDMA (2R)), and(2S)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine(Octyl-CLinDMA (2S)). In addition to these, an ionizable amino lipid canalso be a lipid including a cyclic amine group.

Ionizable lipids can also be the compounds disclosed in InternationalPublication No. WO 2017/075531 A1, incorporated herein by reference inits entirety. For example, the ionizable amino lipids include, but notlimited to:

and any combination thereof.

Ionizable lipids can also be the compounds disclosed in InternationalPublication No. WO 2015/199952 A1, incorporated herein by reference inits entirety. For example, the ionizable amino lipids include, but notlimited to:

and any combination thereof.

g. Nanoparticle Compositions

The lipid composition of a pharmaceutical composition disclosed hereincan include one or more components in addition to those described above.For example, the lipid composition can include one or more permeabilityenhancer molecules, carbohydrates, polymers, surface altering agents(e.g., surfactants), or other components. For example, a permeabilityenhancer molecule can be a molecule described by U.S. Patent ApplicationPublication No. 2005/0222064. Carbohydrates can include simple sugars(e.g., glucose) and polysaccharides (e.g., glycogen and derivatives andanalogs thereof).

A polymer can be included in and/or used to encapsulate or partiallyencapsulate a pharmaceutical composition disclosed herein (e.g., apharmaceutical composition in lipid nanoparticle form). A polymer can bebiodegradable and/or biocompatible. A polymer can be selected from, butis not limited to, polyamines, polyethers, polyamides, polyesters,polycarbamates, polyureas, polycarbonates, polystyrenes, polyimides,polysulfones, polyurethanes, polyacetylenes, polyethylenes,polyethyleneimines, polyisocyanates, polyacrylates, polymethacrylates,polyacrylonitriles, and polyarylates.

The ratio between the lipid composition and the polynucleotide range canbe from about 10:1 to about 60:1 (wt/wt).

In some embodiments, the ratio between the lipid composition and thepolynucleotide can be about 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1,17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1,29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1,41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, 50:1, 51:1, 52:1,53:1, 54:1, 55:1, 56:1, 57:1, 58:1, 59:1 or 60:1 (wt/wt). In someembodiments, the wt/wt ratio of the lipid composition to thepolynucleotide encoding a therapeutic agent is about 20:1 or about 15:1.

In one embodiment, the lipid nanoparticles described herein can comprisepolynucleotides (e.g., mRNA) in a lipid:polynucleotide weight ratio of5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1 or70:1, or a range or any of these ratios such as, but not limited to, 5:1to about 10:1, from about 5:1 to about 15:1, from about 5:1 to about20:1, from about 5:1 to about 25:1, from about 5:1 to about 30:1, fromabout 5:1 to about 35:1, from about 5:1 to about 40:1, from about 5:1 toabout 45:1, from about 5:1 to about 50:1, from about 5:1 to about 55:1,from about 5:1 to about 60:1, from about 5:1 to about 70:1, from about10:1 to about 15:1, from about 10:1 to about 20:1, from about 10:1 toabout 25:1, from about 10:1 to about 30:1, from about 10:1 to about35:1, from about 10:1 to about 40:1, from about 10:1 to about 45:1, fromabout 10:1 to about 50:1, from about 10:1 to about 55:1, from about 10:1to about 60:1, from about 10:1 to about 70:1, from about 15:1 to about20:1, from about 15:1 to about 25:1, from about 15:1 to about 30:1, fromabout 15:1 to about 35:1, from about 15:1 to about 40:1, from about 15:1to about 45:1, from about 15:1 to about 50:1, from about 15:1 to about55:1, from about 15:1 to about 60:1 or from about 15:1 to about 70:1.

In one embodiment, the lipid nanoparticles described herein can comprisethe polynucleotide in a concentration from approximately 0.1 mg/ml to 2mg/ml such as, but not limited to, 0.1 mg/ml, 0.2 mg/ml, 0.3 mg/ml, 0.4mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.7 mg/ml, 0.8 mg/ml, 0.9 mg/ml, 1.0 mg/ml,1.1 mg/ml, 1.2 mg/ml, 1.3 mg/ml, 1.4 mg/ml, 1.5 mg/ml, 1.6 mg/ml, 1.7mg/ml, 1.8 mg/ml, 1.9 mg/ml, 2.0 mg/ml or greater than 2.0 mg/ml.

In some embodiments, the pharmaceutical compositions disclosed hereinare formulated as lipid nanoparticles (LNP). Accordingly, the presentdisclosure also provides nanoparticle compositions comprising (i) alipid composition comprising a delivery agent such as a compound ofFormula (I) or (III) as described herein, and (ii) a polynucleotideencoding a polypeptide of interest. In such nanoparticle composition,the lipid composition disclosed herein can encapsulate thepolynucleotide encoding a polypeptide of interest.

Nanoparticle compositions are typically sized on the order ofmicrometers or smaller and can include a lipid bilayer. Nanoparticlecompositions encompass lipid nanoparticles (LNPs), liposomes (e.g.,lipid vesicles), and lipoplexes. For example, a nanoparticle compositioncan be a liposome having a lipid bilayer with a diameter of 500 nm orless.

Nanoparticle compositions include, for example, lipid nanoparticles(LNPs), liposomes, and lipoplexes. In some embodiments, nanoparticlecompositions are vesicles including one or more lipid bilayers. Incertain embodiments, a nanoparticle composition includes two or moreconcentric bilayers separated by aqueous compartments. Lipid bilayerscan be functionalized and/or crosslinked to one another. Lipid bilayerscan include one or more ligands, proteins, or channels.

In some embodiments, the nanoparticle compositions of the presentdisclosure comprise at least one compound according to Formula (I),(III), (IV), (V), or (VI). For example, the nanoparticle composition caninclude one or more of Compounds 1-147, or one or more of Compounds1-342. Nanoparticle compositions can also include a variety of othercomponents. For example, the nanoparticle composition may include one ormore other lipids in addition to a lipid according to Formula (I), (II),(III), (IV), (V), or (VI), such as (i) at least one phospholipid, (ii)at least one structural lipid, (iii) at least one PEG-lipid, or (iv) anycombination thereof. Inclusion of structural lipid can be optional, forexample when lipids according to formula III are used in the lipidnanoparticle compositions of the invention.

In some embodiments, the nanoparticle composition comprises a compoundof Formula (I), (e.g., Compounds 18, 25, 26 or 48). In some embodiments,the nanoparticle composition comprises a compound of Formula (I) (e.g.,Compounds 18, 25, 26 or 48) and a phospholipid (e.g., DSPC).

In some embodiments, the nanoparticle composition comprises a compoundof Formula (III) (e.g., Compound 236). In some embodiments, thenanoparticle composition comprises a compound of Formula (III) (e.g.,Compound 236) and a phospholipid (e.g., DOPE or DSPC).

In some embodiments, the nanoparticle composition comprises a lipidcomposition consisting or consisting essentially of compound of Formula(I) (e.g., Compounds 18, 25, 26 or 48). In some embodiments, thenanoparticle composition comprises a lipid composition consisting orconsisting essentially of a compound of Formula (I) (e.g., Compounds 18,25, 26 or 48) and a phospholipid (e.g., DSPC).

In some embodiments, the nanoparticle composition comprises a lipidcomposition consisting or consisting essentially of compound of Formula(III) (e.g., Compound 236). In some embodiments, the nanoparticlecomposition comprises a lipid composition consisting or consistingessentially of a compound of Formula (III) (e.g., Compound 236) and aphospholipid (e.g., DOPE or DSPC).

In one embodiment, a lipid nanoparticle comprises an ionizable lipid, astructural lipid, a phospholipid, a PEG-modified lipid, and mRNA. Insome embodiments, the LNP comprises an ionizable lipid, a PEG-modifiedlipid, a sterol and a phospholipid. In some embodiments, the LNP has amolar ratio of about 20-60% ionizable lipid:about 5-25%phospholipid:about 25-55% sterol; and about 0.5-15% PEG-modified lipid.In some embodiments, the LNP comprises a molar ratio of about 50%ionizable lipid, about 1.5% PEG-modified lipid, about 38.5% cholesteroland about 10% phospholipid. In some embodiments, the LNP comprises amolar ratio of about 55% ionizable lipid, about 2.5% PEG lipid, about32.5% cholesterol and about 10% phospholipid. In some embodiments, theionizable lipid is an ionizable amino lipid, the neutral lipid is aphospholipid, and the sterol is a cholesterol. In some embodiments, theLNP has a molar ratio of 50:38.5:10:1.5 of ionizablelipid:cholesterol:DSPC:PEG lipid. In some embodiments, the ionizablelipid is Compound 18 or Compound 236, and the PEG lipid is Compound 428or PEG-DMG.

In some embodiments, the LNP has a molar ratio of 50:38.5:10:1.5 ofCompound 18:Cholesterol:Phospholipid:Compound 428. In some embodiments,the LNP has a molar ratio of 50:38.5:10:1.5 of Compound18:Cholesterol:DSPC:Compound 428. In some embodiments, the LNP has amolar ratio of 50:38.5:10:1.5 of Compound18:Cholesterol:Phospholipid:PEG-DMG. In some embodiments, the LNP has amolar ratio of 50:38.5:10:1.5 of Compound 18:Cholesterol:DSPC:PEG-DMG.

In some embodiments, the LNP has a molar ratio of 50:38.5:10:1.5 ofCompound 236:Cholesterol:Phospholipid:Compound 428. In some embodiments,the LNP has a molar ratio of 50:38.5:10:1.5 of Compound236:Cholesterol:DSPC:Compound 428.

In some embodiments, the LNP has a molar ratio of 40:38.5:20:1.5 ofCompound 18:Cholesterol:Phospholipid:Compound 428. In some embodiments,the LNP has a molar ratio of 40:38.5:20:1.5 of Compound18:Cholesterol:DSPC:Compound 428. In some embodiments, the LNP has amolar ratio of 40:38.5:20:1.5 of Compound18:Cholesterol:Phospholipid:PEG-DMG. In some embodiments, the LNP has amolar ratio of 40:38.5:20:1.5 of Compound 18:Cholesterol:DSPC:PEG-DMG.

In some embodiments, a nanoparticle composition can have the formulationof Compound 18:Phospholipid:Chol:Compound 428 with a mole ratio of50:10:38.5:1.5. In some embodiments, a nanoparticle composition can havethe formulation of Compound 18:DSPC:Chol:Compound 428 with a mole ratioof 50:10:38.5:1.5. In some embodiments, a nanoparticle composition canhave the formulation of Compound 18:Phospholipid:Chol:PEG-DMG with amole ratio of 50:10:38.5:1.5. In some embodiments, a nanoparticlecomposition can have the formulation of Compound 18:DSPC:Chol:PEG-DMGwith a mole ratio of 50:10:38.5:1.5.

In some embodiments, the LNP has a polydispersity value of less than0.4. In some embodiments, the LNP has a net neutral charge at a neutralpH. In some embodiments, the LNP has a mean diameter of 50-150 nm. Insome embodiments, the LNP has a mean diameter of 80-100 nm.

As generally defined herein, the term “lipid” refers to a small moleculethat has hydrophobic or amphiphilic properties. Lipids may be naturallyoccurring or synthetic. Examples of classes of lipids include, but arenot limited to, fats, waxes, sterol-containing metabolites, vitamins,fatty acids, glycerolipids, glycerophospholipids, sphingolipids,saccharolipids, and polyketides, and prenol lipids. In some instances,the amphiphilic properties of some lipids leads them to form liposomes,vesicles, or membranes in aqueous media.

In some embodiments, a lipid nanoparticle (LNP) may comprise anionizable lipid. As used herein, the term “ionizable lipid” has itsordinary meaning in the art and may refer to a lipid comprising one ormore charged moieties. In some embodiments, an ionizable lipid may bepositively charged or negatively charged. An ionizable lipid may bepositively charged, in which case it can be referred to as “cationiclipid”. In certain embodiments, an ionizable lipid molecule may comprisean amine group, and can be referred to as an ionizable amino lipids. Asused herein, a “charged moiety” is a chemical moiety that carries aformal electronic charge, e.g., monovalent (+1, or −1), divalent (+2, or−2), trivalent (+3, or −3), etc. The charged moiety may be anionic(i.e., negatively charged) or cationic (i.e., positively charged).Examples of positively-charged moieties include amine groups (e.g.,primary, secondary, and/or tertiary amines), ammonium groups, pyridiniumgroup, guanidine groups, and imidizolium groups. In a particularembodiment, the charged moieties comprise amine groups. Examples ofnegatively-charged groups or precursors thereof, include carboxylategroups, sulfonate groups, sulfate groups, phosphonate groups, phosphategroups, hydroxyl groups, and the like. The charge of the charged moietymay vary, in some cases, with the environmental conditions, for example,changes in pH may alter the charge of the moiety, and/or cause themoiety to become charged or uncharged. In general, the charge density ofthe molecule may be selected as desired.

It should be understood that the terms “charged” or “charged moiety”does not refer to a “partial negative charge” or “partial positivecharge” on a molecule. The terms “partial negative charge” and “partialpositive charge” are given its ordinary meaning in the art. A “partialnegative charge” may result when a functional group comprises a bondthat becomes polarized such that electron density is pulled toward oneatom of the bond, creating a partial negative charge on the atom. Thoseof ordinary skill in the art will, in general, recognize bonds that canbecome polarized in this way.

In some embodiments, the ionizable lipid is an ionizable amino lipid,sometimes referred to in the art as an “ionizable cationic lipid”. Inone embodiment, the ionizable amino lipid may have a positively chargedhydrophilic head and a hydrophobic tail that are connected via a linkerstructure.

In addition to these, an ionizable lipid may also be a lipid including acyclic amine group.

In one embodiment, the ionizable lipid may be selected from, but notlimited to, a ionizable lipid described in International PublicationNos. WO2013086354 and WO2013116126; the contents of each of which areincorporated herein by reference in their entirety.

In yet another embodiment, the ionizable lipid may be selected from, butnot limited to, formula CLI-CLXXXXII of U.S. Pat. No. 7,404,969; each ofwhich is incorporated herein by reference in their entirety.

In one embodiment, the lipid may be a cleavable lipid such as thosedescribed in International Publication No. WO2012170889, incorporatedherein by reference in its entirety. In one embodiment, the lipid may besynthesized by methods known in the art and/or as described inInternational Publication Nos. WO2013086354; the contents of each ofwhich are incorporated herein by reference in their entirety.

Nanoparticle compositions can be characterized by a variety of methods.For example, microscopy (e.g., transmission electron microscopy orscanning electron microscopy) can be used to examine the morphology andsize distribution of a nanoparticle composition. Dynamic lightscattering or potentiometry (e.g., potentiometric titrations) can beused to measure zeta potentials. Dynamic light scattering can also beutilized to determine particle sizes. Instruments such as the ZetasizerNano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) can alsobe used to measure multiple characteristics of a nanoparticlecomposition, such as particle size, polydispersity index, and zetapotential.

In some embodiments, the nanoparticle composition comprises a lipidcomposition consisting or consisting essentially of compound of Formula(I) (e.g., Compounds 18, 25, 26 or 48). In some embodiments, thenanoparticle composition comprises a lipid composition consisting orconsisting essentially of a compound of Formula (I) (e.g., Compounds 18,25, 26 or 48) and a phospholipid (e.g., DSPC or MSPC).

Nanoparticle compositions can be characterized by a variety of methods.For example, microscopy (e.g., transmission electron microscopy orscanning electron microscopy) can be used to examine the morphology andsize distribution of a nanoparticle composition. Dynamic lightscattering or potentiometry (e.g., potentiometric titrations) can beused to measure zeta potentials. Dynamic light scattering can also beutilized to determine particle sizes. Instruments such as the ZetasizerNano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) can alsobe used to measure multiple characteristics of a nanoparticlecomposition, such as particle size, polydispersity index, and zetapotential.

The size of the nanoparticles can help counter biological reactions suchas, but not limited to, inflammation, or can increase the biologicaleffect of the polynucleotide.

As used herein, “size” or “mean size” in the context of nanoparticlecompositions refers to the mean diameter of a nanoparticle composition.

In one embodiment, the polynucleotide encoding a polypeptide of interestare formulated in lipid nanoparticles having a diameter from about 10 toabout 100 nm such as, but not limited to, about 10 to about 20 nm, about10 to about 30 nm, about 10 to about 40 nm, about 10 to about 50 nm,about 10 to about 60 nm, about 10 to about 70 nm, about 10 to about 80nm, about 10 to about 90 nm, about 20 to about 30 nm, about 20 to about40 nm, about 20 to about 50 nm, about 20 to about 60 nm, about 20 toabout 70 nm, about 20 to about 80 nm, about 20 to about 90 nm, about 20to about 100 nm, about 30 to about 40 nm, about 30 to about 50 nm, about30 to about 60 nm, about 30 to about 70 nm, about 30 to about 80 nm,about 30 to about 90 nm, about 30 to about 100 nm, about 40 to about 50nm, about 40 to about 60 nm, about 40 to about 70 nm, about 40 to about80 nm, about 40 to about 90 nm, about 40 to about 100 nm, about 50 toabout 60 nm, about 50 to about 70 nm, about 50 to about 80 nm, about 50to about 90 nm, about 50 to about 100 nm, about 60 to about 70 nm, about60 to about 80 nm, about 60 to about 90 nm, about 60 to about 100 nm,about 70 to about 80 nm, about 70 to about 90 nm, about 70 to about 100nm, about 80 to about 90 nm, about 80 to about 100 nm and/or about 90 toabout 100 nm.

In one embodiment, the nanoparticles have a diameter from about 10 to500 nm. In one embodiment, the nanoparticle has a diameter greater than100 nm, greater than 150 nm, greater than 200 nm, greater than 250 nm,greater than 300 nm, greater than 350 nm, greater than 400 nm, greaterthan 450 nm, greater than 500 nm, greater than 550 nm, greater than 600nm, greater than 650 nm, greater than 700 nm, greater than 750 nm,greater than 800 nm, greater than 850 nm, greater than 900 nm, greaterthan 950 nm or greater than 1000 nm.

In some embodiments, the largest dimension of a nanoparticle compositionis 1 am or shorter (e.g., 1 am, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm,400 nm, 300 nm, 200 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75 nm, 50 nm, orshorter).

A nanoparticle composition can be relatively homogenous. Apolydispersity index can be used to indicate the homogeneity of ananoparticle composition, e.g., the particle size distribution of thenanoparticle composition. A small (e.g., less than 0.3) polydispersityindex generally indicates a narrow particle size distribution. Ananoparticle composition can have a polydispersity index from about 0 toabout 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08,0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20,0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersityindex of a nanoparticle composition disclosed herein can be from about0.10 to about 0.20.

The zeta potential of a nanoparticle composition can be used to indicatethe electrokinetic potential of the composition. For example, the zetapotential can describe the surface charge of a nanoparticle composition.Nanoparticle compositions with relatively low charges, positive ornegative, are generally desirable, as more highly charged species caninteract undesirably with cells, tissues, and other elements in thebody. In some embodiments, the zeta potential of a nanoparticlecomposition disclosed herein can be from about −10 mV to about +20 mV,from about −10 mV to about +15 mV, from about 10 mV to about +10 mV,from about −10 mV to about +5 mV, from about −10 mV to about 0 mV, fromabout −10 mV to about −5 mV, from about −5 mV to about +20 mV, fromabout −5 mV to about +15 mV, from about −5 mV to about +10 mV, fromabout −5 mV to about +5 mV, from about −5 mV to about 0 mV, from about 0mV to about +20 mV, from about 0 mV to about +15 mV, from about 0 mV toabout +10 mV, from about 0 mV to about +5 mV, from about +5 mV to about+20 mV, from about +5 mV to about +15 mV, or from about +5 mV to about+10 mV.

In some embodiments, the zeta potential of the lipid nanoparticles canbe from about 0 mV to about 100 mV, from about 0 mV to about 90 mV, fromabout 0 mV to about 80 mV, from about 0 mV to about 70 mV, from about 0mV to about 60 mV, from about 0 mV to about 50 mV, from about 0 mV toabout 40 mV, from about 0 mV to about 30 mV, from about 0 mV to about 20mV, from about 0 mV to about 10 mV, from about 10 mV to about 100 mV,from about 10 mV to about 90 mV, from about 10 mV to about 80 mV, fromabout 10 mV to about 70 mV, from about 10 mV to about 60 mV, from about10 mV to about 50 mV, from about 10 mV to about 40 mV, from about 10 mVto about 30 mV, from about 10 mV to about 20 mV, from about 20 mV toabout 100 mV, from about 20 mV to about 90 mV, from about 20 mV to about80 mV, from about 20 mV to about 70 mV, from about 20 mV to about 60 mV,from about 20 mV to about 50 mV, from about 20 mV to about 40 mV, fromabout 20 mV to about 30 mV, from about 30 mV to about 100 mV, from about30 mV to about 90 mV, from about 30 mV to about 80 mV, from about 30 mVto about 70 mV, from about 30 mV to about 60 mV, from about 30 mV toabout 50 mV, from about 30 mV to about 40 mV, from about 40 mV to about100 mV, from about 40 mV to about 90 mV, from about 40 mV to about 80mV, from about 40 mV to about 70 mV, from about 40 mV to about 60 mV,and from about 40 mV to about 50 mV. In some embodiments, the zetapotential of the lipid nanoparticles can be from about 10 mV to about 50mV, from about 15 mV to about 45 mV, from about 20 mV to about 40 mV,and from about 25 mV to about 35 mV. In some embodiments, the zetapotential of the lipid nanoparticles can be about 10 mV, about 20 mV,about 30 mV, about 40 mV, about 50 mV, about 60 mV, about 70 mV, about80 mV, about 90 mV, and about 100 mV.

The term “encapsulation efficiency” of a polynucleotide describes theamount of the polynucleotide that is encapsulated by or otherwiseassociated with a nanoparticle composition after preparation, relativeto the initial amount provided. As used herein, “encapsulation” canrefer to complete, substantial, or partial enclosure, confinement,surrounding, or encasement.

Encapsulation efficiency is desirably high (e.g., close to 100%). Theencapsulation efficiency can be measured, for example, by comparing theamount of the polynucleotide in a solution containing the nanoparticlecomposition before and after breaking up the nanoparticle compositionwith one or more organic solvents or detergents.

Fluorescence can be used to measure the amount of free polynucleotide ina solution. For the nanoparticle compositions described herein, theencapsulation efficiency of a polynucleotide can be at least 50%, forexample 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulationefficiency can be at least 80%. In certain embodiments, theencapsulation efficiency can be at least 90%.

The amount of a polynucleotide present in a pharmaceutical compositiondisclosed herein can depend on multiple factors such as the size of thepolynucleotide, desired target and/or application, or other propertiesof the nanoparticle composition as well as on the properties of thepolynucleotide.

For example, the amount of an mRNA useful in a nanoparticle compositioncan depend on the size (expressed as length, or molecular mass),sequence, and other characteristics of the mRNA. The relative amounts ofa polynucleotide in a nanoparticle composition can also vary.

The relative amounts of the lipid composition and the polynucleotidepresent in a lipid nanoparticle composition of the present disclosurecan be optimized according to considerations of efficacy andtolerability. For compositions including an mRNA as a polynucleotide,the N:P ratio can serve as a useful metric.

As the N:P ratio of a nanoparticle composition controls both expressionand tolerability, nanoparticle compositions with low N:P ratios andstrong expression are desirable. N:P ratios vary according to the ratioof lipids to RNA in a nanoparticle composition.

In general, a lower N:P ratio is preferred. The one or more RNA, lipids,and amounts thereof can be selected to provide an N:P ratio from about2:1 to about 30:1, such as 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1,12:1, 14:1, 16:1, 18:1, 20:1, 22:1, 24:1, 26:1, 28:1, or 30:1. Incertain embodiments, the N:P ratio can be from about 2:1 to about 8:1.In other embodiments, the N:P ratio is from about 5:1 to about 8:1. Incertain embodiments, the N:P ratio is between 5:1 and 6:1. In onespecific aspect, the N:P ratio is about is about 5.67:1.

In addition to providing nanoparticle compositions, the presentdisclosure also provides methods of producing lipid nanoparticlescomprising encapsulating a polynucleotide. Such method comprises usingany of the pharmaceutical compositions disclosed herein and producinglipid nanoparticles in accordance with methods of production of lipidnanoparticles known in the art. See, e.g., Wang et al. (2015) “Deliveryof oligonucleotides with lipid nanoparticles” Adv. Drug Deliv. Rev.87:68-80; Silva et al. (2015) “Delivery Systems for Biopharmaceuticals.Part I: Nanoparticles and Microparticles” Curr. Pharm. Technol. 16:940-954; Naseri et al. (2015) “Solid Lipid Nanoparticles andNanostructured Lipid Carriers: Structure, Preparation and Application”Adv. Pharm. Bull. 5:305-13; Silva et al. (2015) “Lipid nanoparticles forthe delivery of biopharmaceuticals” Curr. Pharm. Biotechnol. 16:291-302,and references cited therein.

Applications Related to Nanoparticles

It has been discovered that the immunomodulatory therapeuticcompositions described herein are superior to current compositions inseveral ways. First, the lipid nanoparticle (LNP) delivery is superiorto other formulations including liposome or protamine based approachesdescribed in the literature and no additional adjuvants are to benecessary. The use of LNPs enables the effective delivery of chemicallymodified or unmodified mRNA compositions. Both modified and unmodifiedLNP formulated mRNA compositions are superior to conventionalcompositions by a significant degree. In some embodiments theimmunomodulatory therapeutic compositions of the invention are superiorto conventional compositions by a factor of at least 10 fold, 20 fold,40 fold, 50 fold, 100 fold, 500 fold or 1,000 fold.

Although attempts have been made to produce functional RNA vaccines,including mRNA vaccines and self-replicating RNA vaccines, thetherapeutic efficacy of these RNA vaccines have not yet been fullyestablished. Quite surprisingly, the inventors have discovered,according to aspects of the invention, a class of formulations fordelivering immunomodulatory therapeutic compositions in vivo thatresults in significantly enhanced, and in many respects synergistic,immune responses including enhanced antigen generation and functionalantibody production with neutralization capability. These results can beachieved even when significantly lower doses of the mRNA areadministered in comparison with mRNA doses used in other classes oflipid based formulations. The formulations of the invention havedemonstrated significant unexpected in vivo immune responses sufficientto establish the efficacy of functional mRNA compositions asimmunomodulatory therapeutic agents. Additionally, self-replicating RNAvaccines rely on viral replication pathways to deliver enough RNA to acell to produce an immunogenic response. The formulations of theinvention do not require viral replication to produce enough protein toresult in a strong immune response. Thus, the mRNA of the invention arenot self-replicating RNA and do not include components necessary forviral replication.

The invention involves, in some aspects, the surprising finding thatlipid nanoparticle (LNP) formulations significantly enhance theeffectiveness of mRNA compositions, including chemically modified andunmodified mRNA immunomodulatory therapeutic compositions. The efficacyof mRNA containing immunomodulatory therapeutic compositions formulatedin LNP was examined in vivo using several distinct tumor antigens. Inaddition to providing an enhanced immune response, the formulations ofthe invention generate a more rapid immune response with fewer doses ofantigen than other compositions tested. The mRNA-LNP formulations of theinvention also produce quantitatively and qualitatively better immuneresponses than compositions formulated in a different carriers.Additionally, the mRNA-LNP formulations of the invention are superior toother compositions even when the dose of mRNA is lower than othercompositions.

The LNP used in the studies described herein has been used previously todeliver siRNA in various animal models as well as in humans. In view ofthe observations made in association with the siRNA delivery of LNPformulations, the fact that LNP is useful in cancer immunomodulatorytherapeutic compositions is quite surprising. It has been observed thattherapeutic delivery of siRNA formulated in LNP causes an undesirableinflammatory response associated with a transient IgM response,typically leading to a reduction in antigen production and a compromisedimmune response. In contrast to the findings observed with siRNA, theLNP-mRNA formulations of the invention are demonstrated herein togenerate enhanced IgG levels, sufficient for prophylactic andtherapeutic methods rather than transient IgM responses.

Pharmaceutical Compositions

The present disclosure includes pharmaceutical compositions comprisingan mRNA or a nanoparticle (e.g., a lipid nanoparticle) described herein,in combination with one or more pharmaceutically acceptable excipient,carrier or diluent. In particular embodiments, the mRNA is present in ananoparticle, e.g., a lipid nanoparticle. In particular embodiments, themRNA or nanoparticle is present in a pharmaceutical composition. Invarious embodiments, the one or more mRNA present in the pharmaceuticalcomposition is encapsulated in a nanoparticle, e.g., a lipidnanoparticle. In particular embodiments, the molar ratio of the firstmRNA to the second mRNA is about 1:50, about 1:25, about 1:10, about1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1,about 4:1, or about 5:1, about 10:1, about 25:1 or about 50:1. Inparticular embodiments, the molar ratio of the first mRNA to the secondmRNA is greater than 1:1.

In some embodiments, a composition described herein comprises an mRNAencoding an antigen of interest (Ag) and an mRNA encoding a polypeptidethat enhances an immune response to the antigen of interest (e.g.,immune potentiator, e.g., STING polypeptide) (IP) wherein the mRNAencoding the antigen of interest (Ag) and the mRNA encoding thepolypeptide that enhances an immune response to the antigen of interest(e.g., immune potentiator, e.g., STING polypeptide)(IP) are formulatedat an Ag:IP mass ratio of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1,10:1 or 20:1 (or alternatively, an IP:Ag mass ratio of 1:1, 1:2, 1:3,1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10 or 1:20). In some embodiments, thecomposition is formulated at an Ag:IP mass ratio of 1:1. 1.25:1, 1.50:1,1.75:1, 2.0:1, 2.25:1, 2.50:1, 2.75:1, 3.0:1, 3.25:1, 3.50:1, 3.75:1,4.0:1, 4.25:1, 4.50:1, 4.75:1 or 5:1 of mRNA encoding the antigen ofinterest to the mRNA encoding the polypeptide that enhances an immune tothe antigen of interest (e.g., immune potentiator, e.g., STINGpolypeptide). In some embodiments, the composition is formulated at amass ratio of 5:1 of mRNA encoding the antigen of interest to the mRNAencoding the polypeptide that enhances an immune to the antigen ofinterest (e.g., immune potentiator, e.g., STING polypeptide) (Ag:IP massratio of 5:1, or alternatively an IP:Ag mass ratio of 1:5). In someembodiments, the composition is formulated at a mass ratio of 10:1 ofmRNA encoding the antigen of interest to the mRNA encoding thepolypeptide that enhances an immune to the antigen of interest (e.g.,immune potentiator, e.g., STING polypeptide) (Ag:IP mass ratio of 10:1,or alternatively an IP:Ag mass ratio of 1:10).

In some embodiments, a composition described herein comprises an mRNAencoding a KRAS activating oncogene mutation peptide and an mRNAencoding a constitutively active human STING polypeptide wherein themRNA encoding the KRAS activating oncogene mutation peptide and the mRNAencoding the constitutively active human STING polypeptide are presentat a KRAS:STING mass ratio of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1,9:1, 10:1 or 20:1, or alternatively a STING:KRAS mass ratio of 1:1, 1:2,1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10 or 1:20. In some embodiments,the mRNAs are present at a KRAS:STING mass ratio of 1:1. 1.25:1, 1.50:1,1.75:1, 2.0:1, 2.25:1, 2.50:1, 2.75:1, 3.0:1, 3.25:1, 3.50:1, 3.75:1,4.0:1, 4.25:1, 4.50:1, 4.75:1 or 5:1 of mRNA encoding the antigen ofinterest to the mRNA encoding the polypeptide that enhances an immune tothe antigen of interest (e.g., immune potentiator, e.g., STINGpolypeptide). In some embodiments, the mRNAs are present at a mass ratioof 5:1 of mRNA encoding the KRAS activating oncogene mutation peptide tothe mRNA encoding the constitutively active human STING polypeptide(KRAS:STING mass ratio of 5:1, or alternatively STING:KRAS mass ratio of1:5). In some embodiments, the mRNAs are present at a mass ratio of 10:1of mRNA encoding the KRAS activating oncogene mutation peptide to themRNA encoding the constitutively active human STING polypeptide(KRAS:STING mass ratio of 10:1, or alternatively STING:KRAS mass ratioof 1:10).

Pharmaceutical compositions may optionally include one or moreadditional active substances, for example, therapeutically and/orprophylactically active substances. Pharmaceutical compositions of thepresent disclosure may be sterile and/or pyrogen-free. Generalconsiderations in the formulation and/or manufacture of pharmaceuticalagents may be found, for example, in Remington: The Science and Practiceof Pharmacy 21^(st) ed., Lippincott Williams & Wilkins, 2005(incorporated herein by reference in its entirety). In particularembodiments, a pharmaceutical composition comprises an mRNA and a lipidnanoparticle, or complexes thereof.

Formulations of the pharmaceutical compositions described herein may beprepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with an excipient and/orone or more other accessory ingredients, and then, if necessary and/ordesirable, dividing, shaping and/or packaging the product into a desiredsingle- or multi-dose unit.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition in accordance with the disclosure will vary,depending upon the identity, size, and/or condition of the subjecttreated and further depending upon the route by which the composition isto be administered. By way of example, the composition may includebetween 0.1% and 100%, e.g., between 0.5% and 70%, between 1% and 30%,between 5% and 80%, or at least 80% (w/w) active ingredient.

The mRNAs of the disclosure can be formulated using one or moreexcipients to: (1) increase stability; (2) increase cell transfection;(3) permit the sustained or delayed release (e.g., from a depotformulation of the mRNA); (4) alter the biodistribution (e.g., targetthe mRNA to specific tissues or cell types); (5) increase thetranslation of a polypeptide encoded by the mRNA in vivo; and/or (6)alter the release profile of a polypeptide encoded by the mRNA in vivo.In addition to traditional excipients such as any and all solvents,dispersion media, diluents, or other liquid vehicles, dispersion orsuspension aids, surface active agents, isotonic agents, thickening oremulsifying agents, preservatives, excipients of the present disclosurecan include, without limitation, lipidoids, liposomes, lipidnanoparticles (e.g., liposomes and micelles), polymers, lipoplexes,core-shell nanoparticles, peptides, proteins, carbohydrates, cellstransfected with mRNAs (e.g., for transplantation into a subject),hyaluronidase, nanoparticle mimics and combinations thereof.Accordingly, the formulations of the disclosure can include one or moreexcipients, each in an amount that together increases the stability ofthe mRNA, increases cell transfection by the mRNA, increases theexpression of a polypeptide encoded by the mRNA, and/or alters therelease profile of a mRNA-encoded polypeptide. Further, the mRNAs of thepresent disclosure may be formulated using self-assembled nucleic acidnanoparticles.

Various excipients for formulating pharmaceutical compositions andtechniques for preparing the composition are known in the art (seeRemington: The Science and Practice of Pharmacy, 21st Edition, A. R.Gennaro, Lippincott, Williams & Wilkins, Baltimore, Md., 2006;incorporated herein by reference in its entirety). The use of aconventional excipient medium may be contemplated within the scope ofthe present disclosure, except insofar as any conventional excipientmedium may be incompatible with a substance or its derivatives, such asby producing any undesirable biological effect or otherwise interactingin a deleterious manner with any other component(s) of thepharmaceutical composition. Excipients may include, for example:antiadherents, antioxidants, binders, coatings, compression aids,disintegrants, dyes (colors), emollients, emulsifiers, fillers(diluents), film formers or coatings, glidants (flow enhancers),lubricants, preservatives, printing inks, sorbents, suspensing ordispersing agents, sweeteners, and waters of hydration. Exemplaryexcipients include, but are not limited to: butylated hydroxytoluene(BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate,croscarmellose, crosslinked polyvinyl pyrrolidone, citric acid,crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropylcellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate,maltitol, mannitol, methionine, methylcellulose, methyl paraben,microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone,povidone, pregelatinized starch, propyl paraben, retinyl palmitate,shellac, silicon dioxide, sodium carboxymethyl cellulose, sodiumcitrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid,sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, andxylitol.

In some embodiments, the formulations described herein may include atleast one pharmaceutically acceptable salt. Examples of pharmaceuticallyacceptable salts that may be included in a formulation of the disclosureinclude, but are not limited to, acid addition salts, alkali or alkalineearth metal salts, mineral or organic acid salts of basic residues suchas amines; alkali or organic salts of acidic residues such as carboxylicacids; and the like. Representative acid addition salts include acetate,acetic acid, adipate, alginate, ascorbate, aspartate, benzenesulfonate,benzene sulfonic acid, benzoate, bisulfate, borate, butyrate,camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate,glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide,hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts,and the like. Representative alkali or alkaline earth metal saltsinclude sodium, lithium, potassium, calcium, magnesium, and the like, aswell as nontoxic ammonium, quaternary ammonium, and amine cations,including, but not limited to ammonium, tetramethylammonium,tetraethylammonium, methylamine, dimethylamine, trimethylamine,triethylamine, ethylamine, and the like.

In some embodiments, the formulations described herein may contain atleast one type of polynucleotide. As a non-limiting example, theformulations may contain 1, 2, 3, 4, 5 or more than 5 mRNAs describedherein. In some embodiments, the formulations described herein maycontain at least one mRNA encoding a polypeptide and at least onenucleic acid sequence such as, but not limited to, an siRNA, an shRNA, asnoRNA, and an miRNA.

Liquid dosage forms for e.g., parenteral administration include, but arenot limited to, pharmaceutically acceptable emulsions, microemulsions,nanoemulsions, solutions, suspensions, syrups, and/or elixirs. Inaddition to active ingredients, liquid dosage forms may comprise inertdiluents commonly used in the art such as, for example, water or othersolvents, solubilizing agents and emulsifiers such as ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,benzyl benzoate, propylene glycol, 1,3-butylene glycol,dimethylformamide, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfurylalcohol, polyethylene glycols and fatty acid esters of sorbitan, andmixtures thereof. Besides inert diluents, oral compositions can includeadjuvants such as wetting agents, emulsifying and/or suspending agents.In certain embodiments for parenteral administration, compositions aremixed with solubilizing agents such as CREMAPHOR®, alcohols, oils,modified oils, glycols, polysorbates, cyclodextrins, polymers, and/orcombinations thereof.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing agents, wetting agents, and/or suspendingagents. Sterile injectable preparations may be sterile injectablesolutions, suspensions, and/or emulsions in nontoxic parenterallyacceptable diluents and/or solvents, for example, as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution, U.S.P., and isotonic sodiumchloride solution. Sterile, fixed oils are conventionally employed as asolvent or suspending medium. For this purpose any bland fixed oil canbe employed including synthetic mono- or diglycerides. Fatty acids suchas oleic acid can be used in the preparation of injectables. Injectableformulations can be sterilized, for example, by filtration through abacterial-retaining filter, and/or by incorporating sterilizing agentsin the form of sterile solid compositions which can be dissolved ordispersed in sterile water or other sterile injectable medium prior touse.

In some embodiments, pharmaceutical compositions including at least onemRNA described herein are administered to mammals (e.g., humans).Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for administration to humans, it will be understood by theskilled artisan that such compositions are generally suitable foradministration to any other animal, e.g., to a non-human mammal.Modification of pharmaceutical compositions suitable for administrationto humans in order to render the compositions suitable foradministration to various animals is well understood, and the ordinarilyskilled veterinary pharmacologist can design and/or perform suchmodification with merely ordinary, if any, experimentation. Subjects towhich administration of the pharmaceutical compositions is contemplatedinclude, but are not limited to, humans and/or other primates; mammals,including commercially relevant mammals such as cattle, pigs, horses,sheep, cats, dogs, mice, and/or rats; and/or birds, includingcommercially relevant birds such as poultry, chickens, ducks, geese,and/or turkeys. In particular embodiments, a subject is provided withtwo or more mRNAs described herein. In particular embodiments, the firstand second mRNAs are provided to the subject at the same time or atdifferent times, e.g., sequentially. In particular embodiments, thefirst and second mRNAs are provided to the subject in the samepharmaceutical composition or formulation, e.g., to facilitate uptake ofboth mRNAs by the same cells.

The present disclosure also includes kits comprising a containercomprising a mRNA encoding a polypeptide that enhances an immuneresponse. In another embodiment, the kit comprises a containercomprising a mRNA encoding a polypeptide that enhances an immuneresponse, as well as one or more additional mRNAs encoding one or moreantigens or interest. In other embodiments, the kit comprises a firstcontainer comprising the mRNA encoding a polypeptide that enhances animmune response and a second container comprising one or more mRNAsencoding one or more antigens of interest. In particular embodiments,the mRNAs for enhancing an immune response and the mRNA(s) encoding anantigen(s) are present in the same or different nanoparticles and/orpharmaceutical compositions. In particular embodiments, the mRNAs arelyophilized, dried, or freeze-dried.

Methods of Enhancing Immune Responses

The disclosure provides a method for enhancing an immune response to anantigen of interest in a subject, e.g., a human subject. In oneembodiment, the method comprises administering to the subject acomposition of the disclosure (or lipid nanoparticle thereof, orpharmaceutical composition thereof) comprising at least one mRNAconstruct encoding: (i) at least one antigen of interest and (ii) apolypeptide that enhances an immune response against the antigen(s) ofinterest, such that an immune response to the antigen(s) of interest isenhanced. In one embodiment, enhancing an immune response comprisesstimulating cytokine production. In another embodiment, enhancing animmune response comprises enhancing cellular immunity (T cellresponses), such as stimulating antigen-specific CD8⁺ T cell activity,stimulating antigen-specific CD4⁺ T cell activity or increasing thepercentage of “effector memory” CD62L^(lo) T cells. In anotherembodiment, enhancing an immune response comprises enhancing humoralimmunity (B cell responses), such as stimulating antigen-specificantibody production.

In one embodiment of the method, the immune potentiator mRNA encodes apolypeptide that stimulates Type I interferon pathway signaling (e.g.,the immune potentiator encodes a polypeptide such as STING, IRF3, IRF7or any of the additional immune potentiators described herein). Invarious other embodiment of the method, the immune potentiator encodes apolypeptide that stimulates NFkB pathway signaling, stimulates aninflammatory response or stimulates dendritic cell development, activityor mobilization. In one embodiment, the method comprises administeringto the subject an mRNA composition that stimulates dendritic celldevelopment, activity or mobilization prior to administering to thesubject an mRNA composition that stimulates Type I interferon pathwaysignaling. For example, the mRNA composition that stimulates dendriticcell development or activity can be administered 1-30 days, e.g., 3days, 5 days, 7 days, 10 days, 14 days, 21 days, 28 days, prior toadministering the mRNA composition that stimulates Type I interferonpathway signaling.

Enhancement of an immune response in a subject against an antigen(s) ofinterest by an immune potenitator of the disclosure can be evaluated bya variety of methods established in the art for assessing immuneresponses, including but not limited to the methods described in theExamples. For example, in various embodiments, enhancement is evaluatedby levels of intracellular staining (ICS) of CD8⁺ cells for IFN-γ orTNF-α, percentage of splenic or peripheral CD8b⁺ cells, or percentage ofsplenic or peripheral “effector memory” CD62L^(lo) cells.

Compositions of the disclosure are administered to the subject at aneffective amount. In general, an effective amount of the compositionwill allow for efficient production of the encoded polypeptide in thecell. Metrics for efficiency may include polypeptide translation(indicated by polypeptide expression), level of mRNA degradation, andimmune response indicators.

Therapeutic Methods

The methods of the disclosure for enhancing an immune response to anantigen(s) of interest in a subject can be used in a variety of clinicalor therapeutic applications. For example, the methods can be used tostimulate anti-tumor immunity in a subject with a tumor. Accordingly, inone aspect, the disclosure pertains to a method of stimulating animmunogenic response to a tumor in a subject in need thereof, the methodcomprising administering to the subject a composition of the disclosure(or lipid nanoparticle thereof, or pharmaceutical composition thereof)comprising at least one mRNA construct encoding: (i) at least one tumorantigen of interest and (ii) a polypeptide that enhances an immuneresponse against the tumor antigen(s) of interest, such that an immuneresponse to the tumor antigen(s) of interest is enhanced. Suitable tumorantigens of interest include those described herein (e.g. tumorneoantigens, including mutant KRAS antigens). In one embodiment of themethod, the subject is administered a mutant KRAS antigen-STING mRNAconstruct encoding a sequence shown in any of SEQ ID NOs: 107-130.

The disclosure also provides methods of treating or preventing a cancerin a subject in need thereof that involve providing or administering atleast one mRNA composition described herein (i.e., an immune potentiatormRNA and an antigen-encoding mRNA, in the same or separate mRNAconstructs) to the subject. In related embodiments, the subject isprovided with or administered a nanoparticle (e.g., a lipidnanoparticle) comprising the mRNA(s). In further related embodiments,the subject is provided with or administered a pharmaceuticalcomposition of the disclosure to the subject. In particular embodiments,the pharmaceutical composition comprises an mRNA(s) encoding an antigenand an immunostimulatory polypeptide as described herein, or itcomprises a nanoparticle comprising the mRNA(s). In particularembodiments, the mRNA(s) is present in a nanoparticle, e.g., a lipidnanoparticle. In particular embodiments, the mRNA(s) or nanoparticle ispresent in a pharmaceutical composition.

In certain embodiments, the subject in need thereof has been diagnosedwith a cancer, or is considered to be at risk of developing a cancer. Insome embodiments, the cancer is liver cancer, colorectal cancer, amelanoma cancer, a pancreatic cancer, a NSCLC, a cervical cancer or ahead or neck cancer. In particular embodiments, the liver cancer ishepatocellular carcinoma. In some embodiments, the colorectal cancer isa primary tumor or a metastasis. In some embodiments, the cancer is ahematopoetic cancer. In some embodiments, the cancer is an acute myeloidleukemia, a chronic myeloid leukemia, a chronic myelomonocytic leukemia,a myelodystrophic syndrome (including refractory anemias and refractorycytopenias) or a myeloproliferative neoplasm or disease (includingpolycythemia vera, essential thrombocytosis and primary myelofibrosis).In other embodiments, the cancer is a blood-based cancer or ahematopoetic cancer. Selectivity for a particular cancer type can beachieved through the combination of use of an appropriate LNPformulation (e.g., targeting specific cell types) in combination withappropriate regulatory site(s) (e.g., microRNAs) engineered into themRNA constructs.

In some embodiments, the mRNA(s), nanoparticle, or pharmaceuticalcomposition is administered to the patient parenterally. In particularembodiments, the subject is a mammal, e.g., a human. In variousembodiments, the subject is provided with an effective amount of themRNA(s).

The methods of treating cancer can further include treatment of thesubject with additional agents that enhance an anti-tumor response inthe subject and/or that are cytotoxic to the tumor (e.g.,chemotherapeutic agents). Suitable therapeutic agents for use incombination therapy include small molecule chemotherapeutic agents,including protein tyrosine kinase inhibitors, as well as biologicalanti-cancer agents, such as anti-cancer antibodies, including but notlimited to those discussed further below. Combination therapy caninclude administering to the subject an immune checkpoint inhibitor toenhance anti-tumor immunity, such as PD-1 inhibitors, PD-L1 inhibitorsand CTLA-4 inhibitors. Other modulators of immune checkpoints may targetOX-40, OX-40L or ICOS. In one embodiment, an agent that modulates animmune checkpoint is an antibody. In another embodiment, an agent thatmodulates an immune checkpoint is a protein or small molecule modulator.In another embodiment, the agent (such as an mRNA) encodes an antibodymodulator of an immune checkpoint. Non-limiting examples of immunecheckpoint inhibitors that can be used in combination therapy includepembrolizumab, alemtuzumab, nivolumab, pidilizumab, ofatumumab,rituximab, MEDI0680 and PDR001, AMP-224, PF-06801591, BGB-A317,REGN2810, SHR-1210, TSR-042, affimer, avelumab (MSB0010718C),atezolizumab (MPDL3280A), durvalumab (MEDI4736), BMS936559, ipilimumab,tremelimumab, AGEN1884, MEDI6469 and MOXR0916.

A pharmaceutical composition including one or more mRNAs of thedisclosure may be administered to a subject by any suitable route. Insome embodiments, compositions of the disclosure are administered by oneor more of a variety of routes, including parenteral (e.g.,subcutaneous, intracutaneous, intravenous, intraperitoneal,intramuscular, intraarticular, intraarterial, intrasynovial,intrasternal, intrathecal, intralesional, or intracranial injection, aswell as any suitable infusion technique), oral, trans- or intra-dermal,interdermal, rectal, intravaginal, topical (e.g. by powders, ointments,creams, gels, lotions, and/or drops), mucosal, nasal, buccal, enteral,vitreal, intratumoral, sublingual, intranasal; by intratrachealinstillation, bronchial instillation, and/or inhalation; as an oralspray and/or powder, nasal spray, and/or aerosol, and/or through aportal vein catheter. In some embodiments, a composition may beadministered intravenously, intramuscularly, intradermally,intra-arterially, intratumorally, subcutaneously, or by inhalation. Insome embodiments, a composition is administered intramuscularly.However, the present disclosure encompasses the delivery of compositionsof the disclosure by any appropriate route taking into considerationlikely advances in the sciences of drug delivery. In general, the mostappropriate route of administration will depend upon a variety offactors including the nature of the pharmaceutical composition includingone or more mRNAs (e.g., its stability in various bodily environmentssuch as the bloodstream and gastrointestinal tract), and the conditionof the patient (e.g., whether the patient is able to tolerate particularroutes of administration).

In certain embodiments, compositions of the disclosure may beadministered at dosage levels sufficient to deliver from about 0.0001mg/kg to about 10 mg/kg, from about 0.001 mg/kg to about 10 mg/kg, fromabout 0.005 mg/kg to about 10 mg/kg, from about 0.01 mg/kg to about 10mg/kg, from about 0.1 mg/kg to about 10 mg/kg, from about 1 mg/kg toabout 10 mg/kg, from about 2 mg/kg to about 10 mg/kg, from about 5 mg/kgto about 10 mg/kg, from about 0.0001 mg/kg to about 5 mg/kg, from about0.001 mg/kg to about 5 mg/kg, from about 0.005 mg/kg to about 5 mg/kg,from about 0.01 mg/kg to about 5 mg/kg, from about 0.1 mg/kg to about 10mg/kg, from about 1 mg/kg to about 5 mg/kg, from about 2 mg/kg to about5 mg/kg, from about 0.0001 mg/kg to about 1 mg/kg, from about 0.001mg/kg to about 1 mg/kg, from about 0.005 mg/kg to about 1 mg/kg, fromabout 0.01 mg/kg to about 1 mg/kg, or from about 0.1 mg/kg to about 1mg/kg in a given dose, where a dose of 1 mg/kg provides 1 mg of mRNA ornanoparticle per 1 kg of subject body weight. In particular embodiments,a dose of about 0.005 mg/kg to about 5 mg/kg of mRNA or nanoparticle ofthe disclosure may be administrated.

A dose may be administered one or more times per day, in the same or adifferent amount, to obtain a desired level of mRNA expression and/oreffect (e.g., a therapeutic effect). The desired dosage may bedelivered, for example, three times a day, two times a day, once a day,every other day, every third day, every week, every two weeks, everythree weeks, or every four weeks. In certain embodiments, the desireddosage may be delivered using multiple administrations (e.g., two,three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen, or more administrations). In some embodiments, asingle dose may be administered, for example, prior to or after asurgical procedure or in the instance of an acute disease, disorder, orcondition. The specific therapeutically effective, prophylacticallyeffective, or otherwise appropriate dose level for any particularpatient will depend upon a variety of factors including the severity andidentify of a disorder being treated, if any; the one or more mRNAsemployed; the specific composition employed; the age, body weight,general health, sex, and diet of the patient; the time ofadministration, route of administration, and rate of excretion of thespecific pharmaceutical composition employed; the duration of thetreatment; drugs used in combination or coincidental with the specificpharmaceutical composition employed; and like factors well known in themedical arts.

The immunomodulatory therapeutic compositions RNA (e.g., mRNA) and lipidnanoparticles of the disclosure may be administered by any route whichresults in a therapeutically effective outcome. These include, but arenot limited, to intradermal, intramuscular, intranasal, and/orsubcutaneous administration. The present disclosure provides methodscomprising administering RNA compositions and lipid nanoparticles of thedisclosure to a subject in need thereof. The exact amount required willvary from subject to subject, depending on the species, age, and generalcondition of the subject, the severity of the disease, the particularcomposition, its mode of administration, its mode of activity, and thelike. RNA compositions and lipid nanoparticles of the disclosure aretypically formulated in dosage unit form for ease of administration anduniformity of dosage. It will be understood, however, that the totaldaily usage of RNA (e.g., mRNA) compositions may be decided by theattending physician within the scope of sound medical judgment. Thespecific therapeutically effective, prophylactically effective, orappropriate imaging dose level for any particular patient will dependupon a variety of factors including the disorder being treated and theseverity of the disorder; the activity of the specific compoundemployed; the specific composition employed; the age, body weight,general health, sex and diet of the patient; the time of administration,route of administration, and rate of excretion of the specific compoundemployed; the duration of the treatment; drugs used in combination orcoincidental with the specific compound employed; and like factors wellknown in the medical arts.

The effective amount of an RNA composition or lipid nanoparticle of thedisclosure, as provided herein, may be as low as 10 μg, administered forexample as a single dose or as two 5 μg doses. In some embodiments, theeffective amount is a total dose of 10 μg-300 μg. For example, theeffective amount may be a total dose of 10 μg, 20 μg, 25 μg, 30 μg, 35μg, 40 μg, 45 μg, 50 μg, 55 μg, 60 μg, 65 μg, 70 μg, 75 μg, 80 μg, 85μg, 90 μg, 95 μg, 100 μg, 110 μg, 120 μg, 130 μg, 140 μg, 150 μg, 160μg, 170 μg, 180 μg, 190 μg or 200 μg, 210 μg, 220 μg, 230 μg, 240 μg,250 μg, 260 μg, 270 μg, 280 μg, 290 μg or 300 μg. In some embodiments,the effective amount is a total dose of 10 μg-300 μg. In someembodiments, the effective amount is a total dose of 30 μg-100 μg or 50μg-200 μg.

In some embodiments, RNA (e.g., mRNA) compositions and lipidnanoparticles may be administered at dosage levels sufficient to deliver0.0001 mg/kg to 100 mg/kg, 0.001 mg/kg to 0.05 mg/kg, 0.005 mg/kg to0.05 mg/kg, 0.001 mg/kg to 0.005 mg/kg, 0.05 mg/kg to 0.5 mg/kg, 0.01mg/kg to 50 mg/kg, 0.1 mg/kg to 40 mg/kg, 0.5 mg/kg to 30 mg/kg, 0.01mg/kg to 10 mg/kg, 0.1 mg/kg to 10 mg/kg, or 1 mg/kg to 25 mg/kg, ofsubject body weight per day, one or more times a day, per week, permonth, etc. to obtain the desired therapeutic, diagnostic, prophylactic,or imaging effect (see e.g., the range of unit doses described inInternational Publication No. WO2013078199, herein incorporated byreference in its entirety). The desired dosage may be delivered threetimes a day, two times a day, once a day, every other day, every thirdday, every week, every two weeks, every three weeks, every four weeks,every 2 months, every three months, every 6 months, etc. In certainembodiments, the desired dosage may be delivered using multipleadministrations (e.g., two, three, four, five, six, seven, eight, nine,ten, eleven, twelve, thirteen, fourteen, or more administrations). Whenmultiple administrations are employed, split dosing regimens such asthose described herein may be used. In exemplary embodiments, RNA (e.g.,mRNA) compositions may be administered at dosage levels sufficient todeliver 0.0005 mg/kg to 0.01 mg/kg, e.g., about 0.0005 mg/kg to about0.0075 mg/kg, e.g., about 0.0005 mg/kg, about 0.001 mg/kg, about 0.002mg/kg, about 0.003 mg/kg, about 0.004 mg/kg or about 0.005 mg/kg.

In some embodiments, RNA (e.g., mRNA) compositions may be administeredonce or twice (or more) at dosage levels sufficient to deliver 0.025mg/kg to 0.250 mg/kg, 0.025 mg/kg to 0.500 mg/kg, 0.025 mg/kg to 0.750mg/kg, or 0.025 mg/kg to 1.0 mg/kg.

In some embodiments, RNA (e.g., mRNA) compositions may be administeredtwice (e.g., Day 0 and Day 7, Day 0 and Day 14, Day 0 and Day 21, Day 0and Day 28, Day 0 and Day 60, Day 0 and Day 90, Day 0 and Day 120, Day 0and Day 150, Day 0 and Day 180, Day 0 and 3 months later, Day 0 and 6months later, Day 0 and 9 months later, Day 0 and 12 months later, Day 0and 18 months later, Day 0 and 2 years later, Day 0 and 5 years later,or Day 0 and 10 years later) at a total dose of or at dosage levelssufficient to deliver a total dose of 0.0100 mg, 0.025 mg, 0.050 mg,0.075 mg, 0.100 mg, 0.125 mg, 0.150 mg, 0.175 mg, 0.200 mg, 0.225 mg,0.250 mg, 0.275 mg, 0.300 mg, 0.325 mg, 0.350 mg, 0.375 mg, 0.400 mg,0.425 mg, 0.450 mg, 0.475 mg, 0.500 mg, 0.525 mg, 0.550 mg, 0.575 mg,0.600 mg, 0.625 mg, 0.650 mg, 0.675 mg, 0.700 mg, 0.725 mg, 0.750 mg,0.775 mg, 0.800 mg, 0.825 mg, 0.850 mg, 0.875 mg, 0.900 mg, 0.925 mg,0.950 mg, 0.975 mg, or 1.0 mg. Higher and lower dosages and frequency ofadministration are encompassed by the present disclosure. For example, aRNA (e.g., mRNA) composition may be administered three or four times.

In some embodiments, RNA (e.g., mRNA) compositions or lipidnanoparticles comprising the same may be administered twice (e.g., Day 0and Day 7, Day 0 and Day 14, Day 0 and Day 21, Day 0 and Day 28, Day 0and Day 60, Day 0 and Day 90, Day 0 and Day 120, Day 0 and Day 150, Day0 and Day 180, Day 0 and 3 months later, Day 0 and 6 months later, Day 0and 9 months later, Day 0 and 12 months later, Day 0 and 18 monthslater, Day 0 and 2 years later, Day 0 and 5 years later, or Day 0 and 10years later) at a total dose of or at dosage levels sufficient todeliver a total dose of 0.010 mg, 0.025 mg, 0.100 mg or 0.400 mg.

In some embodiments, the RNA (e.g., mRNA)composition or lipidnanoparticles comprising the same for use in a method of vaccinating asubject is administered the subject a single dosage of between 10 μg/kgand 400 μg/kg of the nucleic acid vaccine in an effective amount tovaccinate the subject. In some embodiments, the RNA composition or lipidnanoparticles comprising the same for use in a method of vaccinating asubject is administered the subject a single dosage of between 10 μg and400 μg of the nucleic acid vaccine in an effective amount to vaccinatethe subject. In some embodiments, a RNA (e.g., mRNA) composition orlipid nanoparticles comprising the same for use in a method ofvaccinating a subject is administered to the subject as a single dosageof 25-1000 μg (e.g., a single dosage of mRNA encoding an antigen). Insome embodiments, a RNA composition is administered to the subject as asingle dosage of 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500,550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 μg. For example, aRNA composition may be administered to a subject as a single dose of25-100, 25-500, 50-100, 50-500, 50-1000, 100-500, 100-1000, 250-500,250-1000, or 500-1000 μg. In some embodiments, a RNA (e.g., mRNA)composition or lipid nanoparticles comprising the same for use in amethod of vaccinating a subject is administered to the subject as twodosages, the combination of which equals 25-1000 μg of the RNA (e.g.,mRNA) composition.

An RNA (e.g., mRNA) composition or lipid nanoparticles comprising thesame described herein can be formulated into a dosage form describedherein, such as an intranasal, intratracheal, or injectable (e.g.,intravenous, intraocular, intravitreal, intramuscular, intradermal,intracardiac, intraperitoneal, and subcutaneous).

In some embodiments, a pharmaceutical composition of the disclosure maybe administered in combination with another agent, for example, anothertherapeutic agent, a prophylactic agent, and/or a diagnostic agent. By“in combination with,” it is not intended to imply that the agents mustbe administered at the same time and/or formulated for deliverytogether, although these methods of delivery are within the scope of thepresent disclosure. For example, one or more compositions including oneor more different mRNAs may be administered in combination. Compositionscan be administered concurrently with, prior to, or subsequent to, oneor more other desired therapeutics or medical procedures. In general,each agent will be administered at a dose and/or on a time scheduledetermined for that agent. In some embodiments, the present disclosureencompasses the delivery of compositions of the disclosure, or imaging,diagnostic, or prophylactic compositions thereof in combination withagents that improve their bioavailability, reduce and/or modify theirmetabolism, inhibit their excretion, and/or modify their distributionwithin the body.

Exemplary therapeutic agents that may be administered in combinationwith the compositions of the disclosure include, but are not limited to,cytotoxic, chemotherapeutic, and other therapeutic agents. Cytotoxicagents may include, for example, taxol, cytochalasin B, gramicidin D,ethidium bromide, emetine, mitomycin, etoposide, teniposide,vincristine, vinblastine, colchicine, doxorubicin, daunorubicin,dihydroxyanthracinedione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, puromycin, maytansinoids, rachelmycin, and analogs thereof.Radioactive ions may also be used as therapeutic agents and may include,for example, radioactive iodine, strontium, phosphorous, palladium,cesium, iridium, cobalt, yttrium, samarium, and praseodymium. Othertherapeutic agents may include, for example, antimetabolites (e.g.,methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, and5-fluorouracil, and decarbazine), alkylating agents (e.g.,mechlorethamine, thiotepa, chlorambucil, rachelmycin, melphalan,carmustine, lomustine, cyclophosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II)(DDP), and cisplatin), anthracyclines (e.g., daunorubicin anddoxorubicin), antibiotics (e.g., dactinomycin, bleomycin, mithramycin,and anthramycin), and anti-mitotic agents (e.g., vincristine,vinblastine, taxol, and maytansinoids).

The particular combination of therapies (therapeutics or procedures) toemploy in a combination regimen will take into account compatibility ofthe desired therapeutics and/or procedures and the desired therapeuticeffect to be achieved. It will also be appreciated that the therapiesemployed may achieve a desired effect for the same disorder (forexample, a composition useful for treating cancer may be administeredconcurrently with a chemotherapeutic agent), or they may achievedifferent effects (e.g., control of any adverse effects).

Immune checkpoint inhibitors such as pembrolizumab or nivolumab, whichtarget the interaction between programmed death receptor 1/programmeddeath ligand 1 (PD-1/PD-L1) and PD-L2, have been recently approved forthe treatment of various malignancies and are currently beinginvestigated in clinical trials for various cancers including melanoma,head and neck squamous cell carcinoma (HNSCC).

Accordingly, one aspect of the disclosure relates to combination therapyin which a subject is previously treated with a PD-1 antagonist prior toadministration of a lipid nanoparticle or composition of the presentdisclosure. In another aspect, the subject has been treated with amonoclonal antibody that binds to PD-1 prior to administration of alipid nanoparticle or composition of the present disclosure. In anotheraspect, the subject has been administered a lipid nanoparticle orcomposition of the disclosure prior to treatment with an anti-PD-1monoclonal antibody therapy. In some aspects, the anti-PD-1 monoclonalantibody therapy comprises nivolumab, pembrolizumab, pidilizumab, or anycombination thereof. In some aspects, the anti-PD-1 monoclonal antibodycomprises pembrolizumab.

In another aspect, the subject has been treated with a monoclonalantibody that binds to PD-L1 prior to administration of a lipidnanoparticle or composition of the present disclosure. In anotheraspect, the subject is administered a lipid nanoparticle or compositionprior to treatment with an anti-PD-L1 monoclonal antibody therapy. Insome aspects, the anti-PD-L1 monoclonal antibody therapy comprisesdurvalumab, avelumab, MEDI473, BMS-936559, aezolizumab, or anycombination thereof.

In some aspects, the subject has been treated with a CTLA-4 antagonistprior to treatment with the compositions of present disclosure. Inanother aspect, the subject has been previously treated with amonoclonal antibody that binds to CTLA-4 prior to administration of alipid nanoparticle or composition of the present disclosure. In someaspects, the subject has been administered a lipid nanoparticle orcomposition prior to treatment with an anti-CTLA-4 monoclonal antibody.In some aspects, the anti-CTLA-4 antibody therapy comprises ipilimumabor tremelimumab.

In any of the foregoing or related aspects, the disclosure provides alipid nanoparticle, and an optional pharmaceutically acceptable carrier,or a pharmaceutical composition for use in treating or delayingprogression of cancer in an individual, wherein the treatment comprisesadministration of the composition in combination with a secondcomposition, wherein the second composition comprises a checkpointinhibitor polypeptide and an optional pharmaceutically acceptablecarrier.

In any of the foregoing or related aspects, the disclosure provides useof a lipid nanoparticle, and an optional pharmaceutically acceptablecarrier, in the manufacture of a medicament for treating or delayingprogression of cancer in an individual, wherein the medicament comprisesthe lipid nanoparticle and an optional pharmaceutically acceptablecarrier and wherein the treatment comprises administration of themedicament in combination with a composition comprising a checkpointinhibitor polypeptide and an optional pharmaceutically acceptablecarrier.

In any of the foregoing or related aspects, the disclosure provides akit comprising a container comprising a lipid nanoparticle, and anoptional pharmaceutically acceptable carrier, or a pharmaceuticalcomposition, and a package insert comprising instructions foradministration of the lipid nanoparticle or pharmaceutical compositionfor treating or delaying progression of cancer in an individual. In someaspects, the package insert further comprises instructions foradministration of the lipid nanoparticle or pharmaceutical compositionin combination with a composition comprising a checkpoint inhibitorpolypeptide and an optional pharmaceutically acceptable carrier fortreating or delaying progression of cancer in an individual.

In any of the foregoing or related aspects, the disclosure provides akit comprising a medicament comprising a lipid nanoparticle, and anoptional pharmaceutically acceptable carrier, or a pharmaceuticalcomposition, and a package insert comprising instructions foradministration of the medicament alone or in combination with acomposition comprising a checkpoint inhibitor polypeptide and anoptional pharmaceutically acceptable carrier for treating or delayingprogression of cancer in an individual. In some aspects, the kit furthercomprises a package insert comprising instructions for administration ofthe first medicament prior to, current with, or subsequent toadministration of the second medicament for treating or delayingprogression of cancer in an individual.

In any of the foregoing or related aspects, the disclosure provides alipid nanoparticle, a composition, or the use thereof, or a kitcomprising a lipid nanoparticle or a composition as described herein,wherein the checkpoint inhibitor polypeptide inhibits PD1, PD-L1, CTLA4,or a combination thereof. In some aspects, the checkpoint inhibitorpolypeptide is an antibody. In some aspects, the checkpoint inhibitorpolypeptide is an antibody selected from an anti-CTLA4 antibody orantigen-binding fragment thereof that specifically binds CTLA4, ananti-PD1 antibody or antigen-binding fragment thereof that specificallybinds PD1, an anti-PD-L1 antibody or antigen-binding fragment thereofthat specifically binds PD-L1, and a combination thereof. In someaspects, the checkpoint inhibitor polypeptide is an anti-PD-L1 antibodyselected from atezolizumab, avelumab, or durvalumab. In some aspects,the checkpoint inhibitor polypeptide is an anti-CTLA-4 antibody selectedfrom tremelimumab or ipilimumab. In some aspects, the checkpointinhibitor polypeptide is an anti-PD1 antibody selected from nivolumab orpembrolizumab. In some asepcts, the checkpoint inhibitor polypeptide isan anti-PD1 antibody, wherein the anti-PD1 antibody is pembrolizumab.

In related aspects, the disclosure provides a method of reducing ordecreasing a size of a tumor or inhibiting a tumor growth in a subjectin need thereof comprising administering to the subject any of theforegoing or related lipid nanoparticles of the disclosure, or any ofthe foregoing or related compositions of the disclosure.

In related aspects, the disclosure provides a method inducing ananti-tumor response in a subject with cancer comprising administering tothe subject any of the foregoing or related lipid nanoparticles of thedisclosure, or any of the foregoing or related compositions of thedisclosure. In some aspects, the anti-tumor response comprises a T-cellresponse. In some aspects, the T-cell response comprises CD8+ T cells.

In some aspects of the foregoing methods, the method further comprisesadministering a second composition comprising a checkpoint inhibitorpolypeptide, and an optional pharmaceutically acceptable carrier. Insome aspects, the checkpoint inhibitor polypeptide inhibits PD1, PD-L,CTLA4, or a combination thereof. In some aspects, the checkpointinhibitor polypeptide is an antibody. In some aspects, the checkpointinhibitor polypeptide is an antibody selected from an anti-CTLA4antibody or antigen-binding fragment thereof that specifically bindsCTLA4, an anti-PD1 antibody or antigen-binding fragment thereof thatspecifically binds PD1, an anti-PD-L1 antibody or antigen-bindingfragment thereof that specifically binds PD-L1, and a combinationthereof. In some aspects, the checkpoint inhibitor polypeptide is ananti-PD-L1 antibody selected from atezolizumab, avelumab, or durvalumab.In some aspects, the checkpoint inhibitor polypeptide is an anti-CTLA-4antibody selected from tremelimumab or ipilimumab. In some aspects, thecheckpoint inhibitor polypeptide is an anti-PD1 antibody selected fromnivolumab or pembrolizumab. In some asepcts, the checkpoint inhibitorpolypeptide is an anti-PD1 antibody, wherein the anti-PD1 antibody ispembrolizumab.

In some aspects of any of the foregoing or related methods, thecomposition comprising the checkpoint inhibitor polypeptide isadministered by intravenous injection. In some aspects, the compositioncomprising the checkpoint inhibitor polypeptide is administered onceevery 2 to 3 weeks. In some aspects, the composition comprising thecheckpoint inhibitor polypeptide is administered once every 2 weeks oronce every 3 weeks. In some aspects, the composition comprising thecheckpoint inhibitor polypeptide is administered prior to, concurrentwith, or subsequent to administration of the lipid nanoparticle orpharmaceutical composition thereof.

In some aspects of any of the foregoing or related methods, the subjecthas a histologically confirmed KRAS mutation selected from G12D, G12V,G13D or G12C.

In some aspects of any of the foregoing or related methods, the subjecthas metastatic colorectal cancer.

In some aspects of any of the foregoing or related methods, the subjecthas non-small cell lung cancer (NSCLC).

In some aspects of any of the foregoing or related methods, the subjecthas pancreatic cancer.

In any of the foregoing or related aspects, the disclosure providespharmaceutical composition comprising the lipid nanoparticle, and apharmaceutically acceptable carrier. In some aspects, the pharmaceuticalcomposition is formulated for intramuscular delivery.

Other Embodiments of the Disclosure

E1. An immunomodulatory therapeutic composition, comprising:

one or more mRNA each having an open reading frame encoding anactivating oncogene mutation peptide;

one or more mRNA each having an open reading frame encoding apolypeptide that enhances an immune response to the activating oncogenemutation peptide in a subject, wherein the immune response comprises acellular or humoral immune response characterized by:

(i) stimulating Type I interferon pathway signaling,

(ii) stimulating NFkB pathway signaling,

(iii) stimulating an inflammatory response,

(iv) stimulating cytokine production,

(v) stimulating dendritic cell development, activity or mobilization,and

(vi) a combination of any of (i)-(v); and

a pharmaceutically acceptable carrier.

E2. The immunomodulatory therapeutic composition of embodiment 1,wherein the activating oncogene mutation is a KRAS mutation.E3. The immunomodulatory therapeutic composition of embodiment 2,wherein the KRAS mutation is a G12 mutation.E4. The immunomodulatory therapeutic composition of embodiment 3,wherein the G12 KRAS mutation is selected from G12D, G12V, G12S, G12C,G12A, and G12R KRAS mutations.E5. The immunomodulatory therapeutic composition of embodiment 3,wherein the G12 KRAS mutation is selected from G12D, G12V, and G12C KRASmutations.E6. The immunomodulatory therapeutic composition of any one ofembodiments 2-5, wherein the KRAS mutation is a G13 mutation.E7. The immunomodulatory therapeutic composition of embodiment 6,wherein the G13 KRAS mutation is a G13D KRAS mutation.E8. The immunomodulatory therapeutic composition of embodiment 1,wherein the activating oncogene mutation is a H-RAS or N-RAS mutation.E9. The immunomodulatory therapeutic composition of any one ofembodiments 1-8, wherein the mRNA has an open reading frame encoding aconcatemer of two or more activating oncogene mutation peptides.E10. The immunomodulatory therapeutic composition of embodiment 9,wherein the concatemer comprises 3, 4, 5, 6, 7, 8, 9, or 10 activatingoncogene mutation peptides.E11. The immunomodulatory therapeutic composition of embodiment 9,wherein the concatemer comprises 4 activating oncogene mutationpeptides.E12. The immunomodulatory therapeutic composition of embodiment 11,wherein the concatemer comprises KRAS activating oncogene mutationpeptides G12D, G12V, G12C, and G13D.E13. The immunomodulatory therapeutic composition of embodiment 12,wherein the concatemer comprises from N- to C-terminus G12D, G12V, G13D,and G12C.E14. The immunomodulatory therapeutic composition of embodiment 12,wherein the concatemer comprises from N- to C-terminus G12C, G13D, G12V,and G12D.E15. The immunomodulatory therapeutic composition of any one ofembodiments 1-8, wherein the composition comprises 1, 2, 3, or 4 mRNAsencoding 1, 2, 3, or 4 activating oncogene mutation peptides.E16. The immunomodulatory therapeutic composition of embodiment 15,wherein the composition comprises 4 mRNAs encoding 4 activating oncogenemutation peptides.E17. The immunomodulatory therapeutic composition of embodiment 16,wherein the 4 mRNAs encode KRAS activating oncogene mutation peptidesG12D, G12V, G12C, and G13D.E18. The immunomodulatory therapeutic composition of any one ofembodiments 1-17, wherein the activating oncogene mutation peptidecomprises 10-30, 15-25, or 20-25 amino acids in length.E19. The immunomodulatory therapeutic composition of embodiment 18,wherein the activating oncogene mutation peptide comprises 20, 21, 22,23, 24, or 25 amino acids in length.E20. The immunomodulatory therapeutic composition of embodiment 19,wherein the activating oncogene mutation peptide comprises 25 aminoacids in length.E21. The immunomodulatory therapeutic composition of any one ofembodiments 1-20, wherein the mRNA encoding a polypeptide that enhancesan immune response to the activating oncogene mutation peptide in asubject encodes a constitutively active human STING polypeptide.E22. The immunomodulatory therapeutic composition of embodiment 21,wherein the constitutively active human STING polypeptide comprises oneor more mutations selected from the group consisting of V147L, N154S,V155M, R284M, R284K, R284T, E315Q, R375A, and combinations thereof.E23. The immunomodulatory therapeutic composition of embodiment 22,wherein the constitutively active human STING polypeptide comprisesmutation V155M.E24. The immunomodulatory therapeutic composition of embodiment 22,wherein the constitutively active human STING polypeptide comprisesmutations V147L/N154S/V155M.E25. The immunomodulatory therapeutic composition of embodiment 22,wherein the constitutively active human STING polypeptide comprisesmutations R284M/V147L/N154S/V155M.E26. The immunomodulatory therapeutic composition of embodiment 22,wherein the constitutively active human STING polypeptide comprises anamino acid sequence shown in any one of SEQ ID NOs: 1-10 and 164.E27. The immunomodulatory therapeutic composition of any one ofembodiments 21-26, wherein the mRNA encoding the constitutively activehuman STING polypeptide comprises a 3′ UTR comprising at least onemiR-122 microRNA binding site.E28. The immunomodulatory therapeutic composition of any one ofembodiments 1-20, wherein the mRNA encoding a polypeptide that enhancesan immune response to the activating oncogene mutation peptide in asubject encodes a constitutively active human IRF3 polypeptide.E29. The immunomodulatory therapeutic composition of embodiment 28,wherein the constitutively active human IRF3 polypeptide comprises anS396D mutation.E30. The immunomodulatory therapeutic composition of embodiment 28,wherein the constitutively active human IRF3 polypeptide comprises anamino acid sequence shown in SEQ ID NOs: 12.E31. The immunomodulatory therapeutic composition of any one ofembodiments 1-20, wherein the mRNA encoding a polypeptide that enhancesan immune response to the activating oncogene mutation peptide in asubject encodes a constitutively active human IRF7 polypeptide.E32. The immunomodulatory therapeutic composition of embodiment 31,wherein the constitutively active human IRF7 polypeptide comprises oneor more mutations selected from the group consisting of S475D, S476D,S477D, S479D, L480D, S483D, S487D, deletion of amino acids 247-467,deletion of amino acid residues 152-246, deletion of amino acid residues1-151, and combinations thereof.E33. The immunomodulatory therapeutic composition of embodiment 31,wherein the constitutively active human IRF7 polypeptide comprises anamino acid sequence shown in any one of SEQ ID NOs: 14-18.E34. The immunomodulatory therapeutic composition of any one ofembodiments 1-33, wherein the composition further comprises a cancertherapeutic agent.E35. The immunomodulatory therapeutic composition of any one ofembodiments 1-33, wherein the composition further comprises aninhibitory checkpoint polypeptide.E36. The immunomodulatory therapeutic composition of embodiment 35,wherein the inhibitory checkpoint polypeptide is an antibody or fragmentthereof that specifically binds to a molecule selected from the groupconsisting of PD-1, PD-L, TIM-3, VISTA, A2AR, B7-H3, B7-H4, BTLA,CTLA-4, IDO, KIR and LAG3.E37. The immunomodulatory therapeutic composition of any one ofembodiments 1-33, wherein the mRNA is formulated in a lipidnanoparticle.E38. The immunomodulatory therapeutic composition of embodiment 37,wherein the lipid nanoparticle comprises a molar ratio of about 20-60%ionizable amino lipid:5-25% phospholipid:25-55% sterol; and 0.5-15%PEG-modified lipid.E39. The immunomodulatory therapeutic composition of embodiment 38,wherein the ionizable amino lipid is selected from the group consistingof for example, 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane(DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate(DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319).E40. The immunomodulatory therapeutic composition of any one ofembodiments 1-39, wherein each mRNA includes at least one chemicalmodification.E41. The immunomodulatory therapeutic composition of embodiment 40,wherein the chemical modification is selected from the group consistingof pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4′-thiouridine,5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine,2-thio-dihydropseudouridine, 2-thio-dihydrouridine,2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine,4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine,4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine,5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2′-O-methyluridine.E42. An immunomodulatory therapeutic composition, comprising:

one or more mRNA each having an open reading frame encoding a KRASactivating oncogene mutation peptide;

one or more mRNA each having an open reading frame encoding aconstitutively active human STING polypeptide; and

a pharmaceutically acceptable carrier.

E43. The immunomodulatory therapeutic composition of embodiment 42,wherein the constitutively active human STING polypeptide comprisesmutation V155M.E44. The immunomodulatory therapeutic composition of embodiment 43,wherein the constitutively active human STING polypeptide comprises anamino acid sequence shown in SEQ ID NO: 1.E45. The immunomodulatory therapeutic composition of any one ofembodiments 42-44, wherein the mRNA encoding the constitutively activehuman STING polypeptide comprises a 3′ UTR comprising at least onemiR-122 microRNA binding site.E46. The immunomodulatory therapeutic composition of any one ofembodiments 42-45, wherein the KRAS activating oncogene mutation peptideis selected from G12D, G12V, G12S, G12C, G12A, G12R, and G13D.E47. The immunomodulatory therapeutic composition of embodiment 46,wherein the KRAS activating oncogene mutation peptide is selected fromG12D, G12V, G12C, and G13D.E48. The immunomodulatory therapeutic composition of any one ofembodiments 42-47, wherein the mRNA has an open reading frame encoding aconcatemer of two or more KRAS activating oncogene mutation peptides.E49. The immunomodulatory therapeutic composition of embodiment 48,wherein the concatemer comprises 3, 4, 5, 6, 7, 8, 9 or 10 KRASactivating oncogene mutation peptides.E50. The immunomodulatory therapeutic composition of embodiment 49,wherein the concatemer comprises 4 KRAS activating oncogene mutationpeptides.E51. The immunomodulatory therapeutic composition of embodiment 50,wherein the concatemer comprises G12D, G12V, G12C, and G13D.E52. The immunomodulatory therapeutic composition of embodiment 51,wherein the concatemer comprises from N- to C-terminus G12D, G12V, G13D,and G12C.E53. The immunomodulatory therapeutic composition of embodiment 51,wherein the concatemer comprises from N- to C-terminus G12C, G13D, G12V,and G12D.E54. The immunomodulatory therapeutic composition of any one ofembodiments 42-47, wherein the composition comprises 1, 2, 3, or 4 mRNAsencoding 1, 2, 3, or 4 KRAS activating oncogene mutation peptides.E55. The immunomodulatory therapeutic composition of embodiment 54,wherein the composition comprises 4 mRNAs encoding 4 KRAS activatingoncogene mutation peptides.E56. The immunomodulatory therapeutic composition of embodiment 54,wherein the 4 KRAS activating oncogene mutation peptides comprise G12D,G12V, G12C, and G13D.E57. The immunomodulatory therapeutic composition of any one ofembodiments 42-56, wherein the KRAS activating oncogene mutation peptidecomprises 10-30, 15-25, or 20-25 amino acids in length.E58. The immunomodulatory therapeutic composition of embodiment 57,wherein the KRAS activating oncogene mutation peptide comprises 20, 21,22, 23, 24, or 25 amino acids in length.E59. The immunomodulatory therapeutic composition of embodiment 58,wherein the activating oncogene mutation peptide comprises 25 aminoacids in length.E60. The immunomodulatory therapeutic composition of embodiment 51,wherein the concatemer comprises an amino acid sequence selected fromthe group set forth in SEQ ID NOs: 42-47, 73 and 137.E61. The immunomodulatory therapeutic composition of embodiment 51,wherein the mRNA encoding the concatemer comprises the nucleotidesequence selected from the group set forth in SEQ ID NOs: 129-131, 133and 138.E62. The immunomodulatory therapeutic composition of embodiment 54,wherein the KRAS activating oncogene mutation peptides comprise an aminoacid sequence selected from the group set forth in SEQ ID NOs: 36-41, 72and 125.E63. The immunomodulatory therapeutic composition of embodiment 54,wherein the KRAS activating oncogene mutation peptides comprise theamino acid sequence set forth in SEQ ID NOs: 39-41.E64. The immunomodulatory therapeutic composition of embodiment 55,wherein the KRAS activating oncogene mutation peptides comprise theamino acid sequences set forth in SEQ ID NOs: 39-41 and 72.E65. The immunomodulatory therapeutic composition of embodiment 63,wherein the mRNA encoding the KRAS activating oncogene mutation peptidecomprises a nucleotide sequence selected from the group set forth in SEQID NOs: 126-128.E66. The immunomodulatory therapeutic composition of embodiment 64,wherein the mRNA encoding the KRAS activating oncogene mutation peptidecomprises the nucleotide sequences set forth in SEQ ID NOs: 126-128 and132.E67. The immunomodulatory therapeutic composition of any one ofembodiments 42-66, wherein each mRNA is formulated in the same ordifferent lipid nanoparticle.E68. The immunomodulatory therapeutic composition of embodiment 67,wherein each mRNA encoding a KRAS activating oncogene mutation peptideis formulated in the same or different lipid nanoparticle.E69. The immunomodulatory therapeutic composition of embodiment 68,wherein each mRNA encoding constitutively active human STING isformulated in the same or different lipid nanoparticle.E70. The immunomodulatory therapeutic composition of any one ofembodiments 68-69, wherein each mRNA encoding a KRAS activating oncogenemutation peptide is formulated in the same lipid nanoparticle and eachmRNA encoding constitutively active human STING is formulated in adifferent lipid nanoparticle.E71. The immunomodulatory therapeutic composition of any one ofembodiments 68-69, wherein each mRNA encoding a KRAS activating oncogenemutation peptide is formulated in the same lipid nanoparticle and eachmRNA encoding constitutively active human STING is formulated in thesame lipid nanoparticle as each mRNA encoding a KRAS activating oncogenemutation peptide.E72. The immunomodulatory therapeutic composition of any one ofembodiments 68-69, wherein each mRNA encoding a KRAS activating oncogenemutation peptide is formulated in a different lipid nanoparticle andeach mRNA encoding constitutively active human STING is formulated inthe same lipid nanoparticle as each mRNA encoding each KRAS activatingoncogene mutation peptide.E73. The immunomodulatory therapeutic composition of any one ofembodiments 68-72, wherein the lipid nanoparticle comprises a molarratio of about 20-60% ionizable amino lipid:5-25% phospholipid:25-55%sterol; and 0.5-15% PEG-modified lipid.E74. The immunomodulatory therapeutic composition of embodiment 73,wherein the ionizable amino lipid is selected from the group consistingof for example, 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane(DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate(DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319).E75. The immunomodulatory therapeutic composition of any one ofembodiments 42-74, wherein each mRNA includes at least one chemicalmodification.E76. The immunomodulatory therapeutic composition of embodiment 75,wherein the chemical modification is selected from the group consistingof pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4′-thiouridine,5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine,2-thio-dihydropseudouridine, 2-thio-dihydrouridine,2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine,4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine,4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine,5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2′-O-methyluridine.E77. A lipid nanoparticle comprising:

an mRNA having an open reading frame encoding a concatemer of 4 KRASactivating oncogene mutation peptides, wherein the 4 KRAS activatingoncogene mutation peptides comprise G12D, G12V, G12C, and G13D; and

an mRNA having an open reading frame encoding a constitutively activehuman STING polypeptide.

E78. The lipid nanoparticle of embodiment 77, wherein the concatemercomprises from N- to C-terminus G12D, G12V, G13D, and G12C.E79. The lipid nanoparticle of embodiment 77, wherein the concatemercomprises from N- to C-terminus G12C, G13D, G12V, and G12D.E80. The lipid nanoparticle of any one of embodiments 77 to 79, whereineach KRAS activating oncogene mutation peptide comprises 20, 21, 22, 23,24, or 25 amino acids in length.E81. The lipid nanoparticle of embodiment 80, wherein each KRASactivating oncogene mutation peptide comprises 25 amino acids in length.E82. The lipid nanoparticle of embodiment 77, wherein the concatemercomprises an amino acid sequence set forth in SEQ ID NO: 137.E83. The lipid nanoparticle of embodiment 77, wherein the mRNA encodingthe concatemer of 4 KRAS activating oncogene mutation peptides comprisesthe nucleotide sequence set forth in SEQ ID NO: 138.E84. The lipid nanoparticle of any one of embodiments 77-83, wherein theconstitutively active human STING polypeptide comprises mutation V155M.E85. The lipid nanoparticle of embodiment 84, wherein the constitutivelyactive human STING polypeptide comprises the amino acid sequence shownin SEQ ID NO: 1.E86. The lipid nanoparticle of embodiment 84, wherein the mRNA encodingthe constitutively active human STING polypeptide comprises a 3′ UTRcomprising at least one miR-122 microRNA binding site.E87. The lipid nanoparticle of embodiment 84, wherein the mRNA encodingthe constitutively active human STING polypeptide comprises thenucleotide sequence shown in SEQ ID NO: 139.E88. A lipid nanoparticle comprising:

an mRNA having an open reading frame encoding a KRAS activating oncogenemutation peptide comprising G12D;

an mRNA having an open reading frame encoding a KRAS activating oncogenemutation peptide comprising G12V;

an mRNA having an open reading frame encoding a KRAS activating oncogenemutation peptide comprising G12C;

an mRNA having an open reading frame encoding a KRAS activating oncogenemutation peptide comprising G13D; and

an mRNA having an open reading frame encoding a constitutively activehuman STING polypeptide.

E89. The lipid nanoparticle of embodiment 88, wherein each KRASactivating oncogene mutation peptide comprises 20, 21, 22, 23, 24, or 25amino acids in length.E90. The lipid nanoparticle of embodiment 89, wherein each KRASactivating oncogene mutation peptide comprises 25 amino acids in length.E91. The lipid nanoparticle of embodiment 88, wherein the KRASactivating oncogene mutation peptides comprise the amino acid sequencesset forth in SEQ ID NOs: 39-41 and 72.E92. The lipid nanoparticle of embodiment 88, wherein the mRNAs encodingthe KRAS activating oncogene mutation peptides comprise the nucleotidesequences set forth in SEQ ID NOs: 126-128 and 132.E93. The lipid nanoparticle of any one of embodiments 88-92, wherein theconstitutively active human STING polypeptide comprises mutation V155M.E94. The lipid nanoparticle of embodiment 93, wherein the constitutivelyactive human STING polypeptide comprises the amino acid sequence shownin SEQ ID NO: 1.E95. The lipid nanoparticle of embodiment 94, wherein the mRNA encodingthe constitutively active human STING polypeptide comprises a 3′ UTRcomprising at least one miR-122 microRNA binding site.E96. The lipid nanoparticle of embodiment 94, wherein the mRNA encodingthe constitutively active human STING polypeptide comprises thenucleotide sequence shown in SEQ ID NO: 139.E97. A lipid nanoparticle comprising:

an mRNA having an open reading frame encoding a KRAS activating oncogenemutation peptide comprising G12D; and

an mRNA having an open reading frame encoding a constitutively activehuman STING polypeptide.

E98. A lipid nanoparticle comprising:

an mRNA having an open reading frame encoding a KRAS activating oncogenemutation peptide comprising G12V; and

an mRNA having an open reading frame encoding a constitutively activehuman STING polypeptide.

E99. A lipid nanoparticle comprising:

an mRNA having an open reading frame encoding a KRAS activating oncogenemutation peptide comprising G12C; and

an mRNA having an open reading frame encoding a constitutively activehuman STING polypeptide.

E100. A lipid nanoparticle comprising:

an mRNA having an open reading frame encoding a KRAS activating oncogenemutation peptide comprising G13D; and

an mRNA having an open reading frame encoding a constitutively activehuman STING polypeptide.

E101. The lipid nanoparticle of any one of embodiments 97-100, whereineach KRAS activating oncogene mutation peptide comprises 20, 21, 22, 23,24, or 25 amino acids in length.E102. The lipid nanoparticle of embodiment 101, wherein each KRASactivating oncogene mutation peptide comprises 25 amino acids in length.E103. The lipid nanoparticle of embodiment 97, wherein the KRASactivating oncogene mutation peptide comprises the amino acid sequenceset forth in SEQ ID NO: 39.E104. The lipid nanoparticle of embodiment 97, wherein the mRNA encodingthe KRAS activating oncogene mutation peptide comprises the nucleotidesequence set forth in SEQ ID NOs: 126.E105. The lipid nanoparticle of embodiment 98, wherein the KRASactivating oncogene mutation peptide comprises the amino acid sequenceset forth in SEQ ID NO:40.E106. The lipid nanoparticle of embodiment 98, wherein the mRNA encodingthe KRAS activating oncogene mutation peptide comprises the nucleotidesequence set forth in SEQ ID NOs: 127.E107. The lipid nanoparticle of embodiment 99, wherein the KRASactivating oncogene mutation peptide comprises the amino acid sequenceset forth in SEQ ID NO: 72.E108. The lipid nanoparticle of embodiment 99, wherein the mRNA encodingthe KRAS activating oncogene mutation peptide comprises the nucleotidesequence set forth in SEQ ID NO: 132.E109. The lipid nanoparticle of embodiment 100, wherein the KRASactivating oncogene mutation peptide comprises the amino acid sequenceset forth in SEQ ID NO: 41.E110. The lipid nanoparticle of embodiment 100, wherein the mRNAencoding the KRAS activating oncogene mutation peptide comprises thenucleotide sequence set forth in SEQ ID NO: 128.E111. The lipid nanoparticle of any one of embodiments 97-110, whereinthe constitutively active human STING polypeptide comprises mutationV155M.E112. The lipid nanoparticle of embodiment 111, wherein theconstitutively active human STING polypeptide comprises the amino acidsequence shown in SEQ ID NO: 1.E113. The lipid nanoparticle of embodiment 111, wherein the mRNAencoding the constitutively active human STING polypeptide comprises a3′ UTR comprising at least one miR-122 microRNA binding site.E114. The lipid nanoparticle of embodiment 111, wherein the mRNAencoding the constitutively active human STING polypeptide comprises thenucleotide sequence shown in SEQ ID NO: 139.E115. A method for treating a subject, comprising:administering to a subject having cancer the immunomodulatorytherapeutic composition of any one of embodiments 1-76 or the lipidnanoparticle of any one of embodiments 77-114.E116. The method of embodiment 115, wherein immunomodulatory therapeuticcomposition or lipid nanoparticle is administered in combination with acancer therapeutic agent.E117. The method of embodiment 115 or 116, wherein immunomodulatorytherapeutic composition or lipid nanoparticle is administered incombination with an inhibitory checkpoint polypeptide.E118. The method of embodiment 117, wherein the inhibitory checkpointpolypeptide is an antibody or fragment thereof that specifically bindsto a molecule selected from the group consisting of PD-1, PD-L1, TIM-3,VISTA, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR and LAG3.E119. The method of any one of embodiments 115-118, wherein the canceris selected from cancer of the pancreas, peritoneum, large intestine,small intestine, biliary tract, lung, endometrium, ovary, genital tract,gastrointestinal tract, cervix, stomach, urinary tract, colon, rectum,and hematopoietic and lymphoid tissues.E120. The method of embodiment 113, wherein the cancer is colorectalcancer.E121. A lipid nanoparticle comprising:

a first mRNA having an open reading frame encoding a concatemer of 4KRAS activating oncogene mutation peptides, wherein the 4 KRASactivating oncogene mutation peptides comprise G12D, G12V, G12C, andG13D; and

a second mRNA having an open reading frame encoding a constitutivelyactive human STING polypeptide,

wherein the first mRNA and second mRNA are present at a KRAS:STING massratio selected from the group consisting of 1:1, 2:1, 3:1, 4:1, 5:1,6:1, 7:1, 8:1, 9:1 or 10:1.

E122. The lipid nanoparticle of embodiment 121, wherein the concatemercomprises from N- to C-terminus G12D, G12V, G13D, and G12C.E123. The lipid nanoparticle of embodiment 121, wherein the concatemercomprises from N- to C-terminus G12C, G13D, G12V, and G12D.E124. The lipid nanoparticle of any one of embodiments 121 to 123,wherein each KRAS activating oncogene mutation peptide comprises 20, 21,22, 23, 24, or 25 amino acids in length.E125. The lipid nanoparticle of embodiment 124, wherein each KRASactivating oncogene mutation peptide comprises 25 amino acids in length.E126. The lipid nanoparticle of embodiment 121, wherein the concatemercomprises an amino acid sequence set forth in SEQ ID NO: 137.E127. The lipid nanoparticle of embodiment 121, wherein the mRNAencoding the concatemer of 4 KRAS activating oncogene mutation peptidescomprises the nucleotide sequence set forth in SEQ ID NO: 138.E128. The lipid nanoparticle of any one of embodiments 121-127, whereinthe constitutively active human STING polypeptide comprises mutationV155M.E129. The lipid nanoparticle of embodiment 128, wherein theconstitutively active human STING polypeptide comprises the amino acidsequence shown in SEQ ID NO: 1.E130. The lipid nanoparticle of embodiment 128, wherein the mRNAencoding the constitutively active human STING polypeptide comprises a3′ UTR comprising at least one miR-122 microRNA binding site.E131. The lipid nanoparticle of embodiment 128, wherein the mRNAencoding the constitutively active human STING polypeptide comprises thenucleotide sequence shown in SEQ ID NO: 139.E132. A lipid nanoparticle comprising:

a first mRNA having an open reading frame encoding a KRAS activatingoncogene mutation peptide comprising G12D;

a second mRNA having an open reading frame encoding a KRAS activatingoncogene mutation peptide comprising G12V;

a third mRNA having an open reading frame encoding a KRAS activatingoncogene mutation peptide comprising G12C;

a fourth mRNA having an open reading frame encoding a KRAS activatingoncogene mutation peptide comprising G13D; and

a fifth mRNA having an open reading frame encoding a constitutivelyactive human STING polypeptide,

wherein the first, second, third, fourth and fifth mRNAs are present ata KRAS:STING mass ratio selected from the group consisting of 1:1, 2:1,3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1.

E133. The lipid nanoparticle of embodiment 132, wherein each KRASactivating oncogene mutation peptide comprises 20, 21, 22, 23, 24, or 25amino acids in length.E134. The lipid nanoparticle of embodiment 133, wherein each KRASactivating oncogene mutation peptide comprises 25 amino acids in length.E135. The lipid nanoparticle of embodiment 132, wherein the KRASactivating oncogene mutation peptides comprise the amino acid sequencesset forth in SEQ ID NOs: 39-41 and 72.E136. The lipid nanoparticle of embodiment 132, wherein the mRNAsencoding the KRAS activating oncogene mutation peptides comprise thenucleotide sequences set forth in SEQ ID NOs: 126-128 and 132.E137. The lipid nanoparticle of any one of embodiments 132-136, whereinthe constitutively active human STING polypeptide comprises mutationV155M.E138. The lipid nanoparticle of embodiment 137, wherein theconstitutively active human STING polypeptide comprises the amino acidsequence shown in SEQ ID NO: 1.E139. The lipid nanoparticle of embodiment 138, wherein the mRNAencoding the constitutively active human STING polypeptide comprises a3′ UTR comprising at least one miR-122 microRNA binding site.E140. The lipid nanoparticle of embodiment 137, wherein the mRNAencoding the constitutively active human STING polypeptide comprises thenucleotide sequence shown in SEQ ID NO: 139.E141. The lipid nanoparticle of any one of embodiments 121-131, whereinthe first and second mRNAs are present at a KRAS:STING mass ratio of1:1.E142. The lipid nanoparticle of any one of embodiments 121-131, whereinthe first and second mRNAs are present at a KRAS:STING mass ratio of2:1.E143. The lipid nanoparticle of any one of embodiments 121-131, whereinthe first and second mRNAs are present at a KRAS:STING mass ratio of3:1.E144. The lipid nanoparticle of any one of embodiments 121-131, whereinthe first and second mRNAs are present at a KRAS:STING mass ratio of4:1.E145. The lipid nanoparticle of any one of embodiments 121-131, whereinthe first and second mRNAs are present at a KRAS:STING mass ratio of5:1.E146. The lipid nanoparticle of any one of embodiments 121-131, whereinthe first and second mRNAs are present KRAS:STING mass ratio of 6:1.E147. The lipid nanoparticle of any one of embodiments 121-131, whereinthe first and second mRNAs are present at a KRAS:STING mass ratio of7:1.E148. The lipid nanoparticle of any one of embodiments 121-131, whereinthe first and second mRNAs are present at a KRAS:STING mass ratio of8:1.E149. The lipid nanoparticle of any one of embodiments 121-140, whereinthe first and second mRNAs are present at a KRAS:STING mass ratio of9:1.E150. The lipid nanoparticle of any one of embodiments 121-140, whereinthe first and second mRNAs are present at a KRAS:STING mass ratio of10:1.E151. A composition comprising:

(i) a first mRNA having an open reading frame encoding a concatemer of 4KRAS activating oncogene mutation peptides, wherein the concatemercomprises from N- to C-terminus G12D, G12V, G13D, and G12C, and

(ii) a second mRNA having an open reading frame encoding aconstitutively active human STING polypeptide, wherein theconstitutively active human STING polypeptide comprises mutation V155M,

wherein the first mRNA and second mRNA are present at a KRAS:STING massratio selected from the group consisting of 1:1, 2:1, 3:1, 4:1, 5:1,6:1, 7:1, 8:1, 9:1 or 10:1;

and a pharmaceutically acceptable carrier.

E152. The composition of embodiment 151, wherein the concatemer of 4KRAS activating oncogene mutation peptides comprises the amino acidsequence set forth in SEQ ID NO: 137.E153. The composition of embodiment 151 or 152, wherein the first mRNAencoding the concatemer of 4 KRAS activating oncogene mutation peptidescomprises the nucleotide sequence set forth in SEQ ID NO: 169.E154. The composition of any one of embodiments 151-153, wherein theconstitutively active human STING polypeptide comprises the amino acidsequence shown in SEQ ID NO: 1.E155. The composition of any one of embodiments 151-154, wherein themRNA encoding the constitutively active human STING polypeptidecomprises the nucleotide sequence shown in SEQ ID NO: 170.E156. The composition of any one of embodiments 151-155, wherein thefirst mRNA comprises a 5′ UTR comprising the nucleotide sequence setforth in SEQ ID NO: 176.E157. The composition of any one of embodiments 151-155, wherein thesecond mRNA comprises a 5′ UTR comprising the nucleotide sequence setforth in SEQ ID NO: 176.E158. The composition of any one of embodiments 151-157, wherein thesecond mRNA encoding the constitutively active human STING polypeptidecomprises a 3′ UTR having a miR-122 microRNA binding site.E159. The composition of embodiment 158, wherein the miR-122 microRNAbinding site comprises the nucleotide sequence shown in SEQ ID NO: 175.E160. The composition of any one of embodiments 151-159, wherein thefirst mRNA and second mRNA each comprise a poly A tail.E161. The composition of embodiment 160, wherein the poly A tailcomprises about 100 nucleotides.E162. The composition of any one of embodiments 151-161, wherein thefirst and second mRNAs each comprise a 5′ Cap 1 structure.E163. The composition of any one of embodiments 151-162, wherein thefirst and second mRNAs each comprise at least one chemical modification.E164. The composition of embodiment 163, wherein the chemicalmodification is N1-methylpseudouridine.E165. The composition of embodiment 164, wherein the first mRNA is fullymodified with N1-methylpseudouridine.E166. The composition of embodiment 164, wherein the second mRNA isfully modified with N1-methylpseudouridine.E167. The composition of any one of embodiments 151-166, wherein thepharmaceutically acceptable carrier comprises a buffer solution.E168. A composition comprising:

(i) a first mRNA comprising the nucleotide sequence set forth in SEQ IDNO: 167, and

(ii) a second mRNA comprising the nucleotide sequence set forth in SEQID NO: 168,

wherein the first and second mRNA are each fully modified withN1-methylpseudouridine, and

wherein the first mRNA and second mRNA are present at a KRAS:STING massratio selected from the group consisting of 1:1, 2:1, 3:1, 4:1, 5:1,6:1, 7:1, 8:1, 9:1 or 10:1;

and a pharmaceutically acceptable carrier.

E169. The composition of embodiment 168, wherein the pharmaceuticallyacceptable carrier comprises a buffer solution.E170. The composition of any one of embodiments 151-169, wherein thefirst and second mRNAs are present at a KRAS:STING mass ratio of 1:1.E171. The composition of any one of embodiments 151-169, wherein thefirst and second mRNAs are present at a KRAS:STING mass ratio of 2:1.E172. The composition of any one of embodiments 151-169, wherein thefirst and second mRNAs are present at a KRAS:STING mass ratio of 3:1.E173. The composition of any one of embodiments 151-169, wherein thefirst and second mRNAs are present at a KRAS:STING mass ratio of 4:1.E174. The composition of any one of embodiments 151-169, wherein thefirst and second mRNAs are present at a KRAS:STING mass ratio of 5:1.E175. The composition of any one of embodiments 151-169, wherein thefirst and second mRNAs are present KRAS:STING mass ratio of 6:1.E176. The composition of any one of embodiments 151-169, wherein thefirst and second mRNAs are present at a KRAS:STING mass ratio of 7:1.E177. The composition of any one of embodiments 151-169, wherein thefirst and second mRNAs are present at a KRAS:STING mass ratio of 8:1.E178. The composition of any one of embodiments 151-169, wherein thefirst and second mRNAs are present at a KRAS:STING mass ratio of 9:1.E179. The composition of any one of embodiments 151-169, wherein thefirst and second mRNAs are present at a KRAS:STING mass ratio of 10:1.E180. The composition of any one of embodiments 151-179, which isformulated in a lipid nanoparticle.E181. The composition of embodiment 180, wherein the lipid nanoparticlecomprises a molar ratio of about 20-60% ionizable amino lipid:5-25%phospholipid:25-55% sterol; and 0.5-15% PEG-modified lipid.E182. The composition of embodiment 181, wherein the lipid nanoparticlecomprises a molar ratio of about 50% Compound 25:about 10% DSPC:about38.5% cholesterol; and about 1.5% PEG-DMG.E183. The composition of any one of embodiments 151-182, which isformulated for intramuscular delivery.E184. A lipid nanoparticle comprising:

(i) a first mRNA having an open reading frame encoding a concatemer of 4KRAS activating oncogene mutation peptides, wherein the concatemercomprises from N- to C-terminus G12D, G12V, G13D, and G12C; and

(ii) a second mRNA having an open reading frame encoding aconstitutively active human STING polypeptide, wherein theconstitutively active human STING polypeptide comprises mutation V155M,

wherein the first mRNA and second mRNA are present at a KRAS:STING massratio of 5:1.

E185. The lipid nanoparticle of embodiment 184, wherein the concatemerof 4 KRAS activating oncogene mutation peptides comprises the amino acidsequence set forth in SEQ ID NO: 137.E186. The lipid nanoparticle of embodiment 184 or 185, wherein the firstmRNA encoding the concatemer of 4 KRAS activating oncogene mutationpeptides comprises the nucleotide sequence set forth in SEQ ID NO: 169.E187. The lipid nanoparticle of any one of embodiments 184-186, whereinthe constitutively active human STING polypeptide comprises the aminoacid sequence shown in SEQ ID NO: 1.E188. The lipid nanoparticle of any one of embodiments 184-187, whereinthe mRNA encoding the constitutively active human STING polypeptidecomprises the nucleotide sequence shown in SEQ ID NO: 170.E189. The lipid nanoparticle of any one of embodiments 184-188, whereinthe first mRNA comprises a 5′ UTR comprising the nucleotide sequenceshown in SEQ ID NO: 176.E190. The lipid nanoparticle of any one of embodiments 184-188, whereinthe second mRNA comprises a 5′ UTR comprising the nucleotide sequenceshown in SEQ ID NO: 176.E191. The lipid nanoparticle of any one of embodiments 184-190, whereinthe second mRNA encoding the constitutively active human STINGpolypeptide comprises a 3′ UTR having a miR-122 microRNA binding site.E192. The lipid nanoparticle of embodiment 191, wherein the miR-122microRNA binding site comprises the nucleotide sequence shown in SEQ IDNO: 175.E193. The lipid nanoparticle of any one of embodiments 184-192, whereinthe first and second mRNAs each comprise a poly A tail.E194. The lipid nanoparticle of embodiment 193, wherein the poly A tailcomprises about 100 nucleotides.E195. The lipid nanoparticle of any one of embodiments 184-194, whereinthe first and second mRNAs each comprise a 5′ Cap 1 structure.E196. The lipid nanoparticle of any one of embodiments 184-195, whereinthe first and second mRNAs each comprise at least one chemicalmodification.E197. The lipid nanoparticle of embodiment 196, wherein the chemicalmodification is N1-methylpseudouridine.E198. The lipid nanoparticle of embodiment 197, wherein the first mRNAis fully modified with N1-methylpseudouridine.E199. The lipid nanoparticle of embodiment 197, wherein the second mRNAis fully modified with N1-methylpseudouridine.E200. A lipid nanoparticle comprising:

(i) a first mRNA comprising the nucleotide sequence set forth in SEQ IDNO: 167; and

(ii) a second mRNA comprising the nucleotide sequence set forth in SEQID NO: 168,

wherein the first and second mRNA are each fully modified withN1-methylpseudouridine, and

wherein the first mRNA and second mRNA are present at a KRAS:STING massratio of 5:1.

E201. The lipid nanoparticle of any one of embodiments 184-200, whereinthe lipid nanoparticle comprises a molar ratio of about 20-60% ionizableamino lipid:5-25% phospholipid:25-55% sterol; and 0.5-15% PEG-modifiedlipid.E202. The lipid nanoparticle of embodiment 201, wherein the lipidnanoparticle comprises a molar ratio of about 50% Compound 25:about 10%DSPC:about 38.5% cholesterol; and about 1.5% PEG-DMG.E203. A pharmaceutical composition comprising the lipid nanoparticle ofany one of embodiments 184-202, and a pharmaceutically acceptablecarrier.E204. The pharmaceutical composition of embodiment 203 which isformulated for intramuscular delivery.E205. The lipid nanoparticle of any one of embodiments 184-202, and anoptional pharmaceutically acceptable carrier, or the pharmaceuticalcomposition of any one of embodiments 203-204 for use in treating ordelaying progression of cancer in an individual, wherein the treatmentcomprises administration of the composition in combination with a secondcomposition, wherein the second composition comprises a checkpointinhibitor polypeptide and an optional pharmaceutically acceptablecarrier.E206. Use of a lipid nanoparticle of any one of embodiments 184-202, andan optional pharmaceutically acceptable carrier, in the manufacture of amedicament for treating or delaying progression of cancer in anindividual, wherein the medicament comprises the lipid nanoparticle andan optional pharmaceutically acceptable carrier and wherein thetreatment comprises administration of the medicament in combination witha composition comprising a checkpoint inhibitor polypeptide and anoptional pharmaceutically acceptable carrier.E207. A kit comprising a container comprising the lipid nanoparticle ofany one of embodiments 184-202, and an optional pharmaceuticallyacceptable carrier, or the pharmaceutical composition of any one ofembodiments 203-204, and a package insert comprising instructions foradministration of the lipid nanoparticle or pharmaceutical compositionfor treating or delaying progression of cancer in an individual.E208. The kit of embodiment 207, wherein the package insert furthercomprises instructions for administration of the lipid nanoparticle orpharmaceutical composition in combination with a composition comprisinga checkpoint inhibitor polypeptide and an optional pharmaceuticallyacceptable carrier for treating or delaying progression of cancer in anindividual.E209. A kit comprising a medicament comprising a lipid nanoparticle ofany one of embodiments 184-202, and an optional pharmaceuticallyacceptable carrier, or the pharmaceutical composition of any one ofembodiments 203-204, and a package insert comprising instructions foradministration of the medicament alone or in combination with acomposition comprising a checkpoint inhibitor polypeptide and anoptional pharmaceutically acceptable carrier for treating or delayingprogression of cancer in an individual.E210. The kit of embodiment 209, wherein the kit further comprises apackage insert comprising instructions for administration of the firstmedicament prior to, current with, or subsequent to administration ofthe second medicament for treating or delaying progression of cancer inan individual.E211. The lipid nanoparticle of any one of embodiments 184-202, thecomposition of any one of embodiments 203-204, the use of embodiments205-206 or the kit of any one of embodiments 207-210, wherein thecheckpoint inhibitor polypeptide inhibits PD1, PD-L1, CTLA4, or acombination thereof.E212. The lipid nanoparticle of any one of embodiments 184-202, thecomposition of embodiments 203-204, the use of embodiment 205-206 or thekit of any one of embodiments 207-210, wherein the checkpoint inhibitorpolypeptide is an antibody.E213. The lipid nanoparticle of any one of embodiments 184-202, thecomposition of embodiments 203-204, the use of embodiment 205-206 or thekit of any one of embodiments 207-210, wherein the checkpoint inhibitorpolypeptide is an antibody selected from an anti-CTLA4 antibody orantigen-binding fragment thereof that specifically binds CTLA4, ananti-PD1 antibody or antigen-binding fragment thereof that specificallybinds PD1, an anti-PD-L1 antibody or antigen-binding fragment thereofthat specifically binds PD-L1, and a combination thereof.E214. The lipid nanoparticle of any one of embodiments 184-202, thecomposition of embodiments 203-204, the use of embodiment 205-206 or thekit of any one of embodiments 207-210, wherein the checkpoint inhibitorpolypeptide is an anti-PD-L1 antibody selected from atezolizumab,avelumab, or durvalumab.E215. The lipid nanoparticle of any one of embodiments 184-202, thecomposition of embodiments 203-204, the use of embodiment 205-206 or thekit of any one of embodiments 197-200, wherein the checkpoint inhibitorpolypeptide is an anti-CTLA-4 antibody selected from tremelimumab oripilimumab.E216. The lipid nanoparticle of any one of embodiments 184-202, thecomposition of embodiments 203-204, the use of embodiment 205-206 or thekit of any one of embodiments 197-200, wherein the checkpoint inhibitorpolypeptide is an anti-PD1 antibody selected from nivolumab orpembrolizumab.E217. A method of reducing or decreasing a size of a tumor or inhibitinga tumor growth in a subject in need thereof comprising administering tothe subject the lipid nanoparticle of any one of embodiments 184-202 orthe composition of any one of embodiments 203-204.E218. A method of inducing an anti-tumor response in a subject withcancer, comprising administering to the subject the lipid nanoparticleof any one of embodiments 184-202 or the composition of any one ofembodiments 203-204.E219. The method of embodiment 218, wherein the anti-tumor responsecomprises a T-cell response.E220. The method of embodiment 219, wherein the T-cell responsecomprises CD8+ T cells.E221. The method of any one of embodiments 217-220, wherein thecomposition is administered by intramuscular injection.E222. The method of any one of embodiments 217-220, further comprisingadministering a second composition comprising a checkpoint inhibitorpolypeptide, and an optional pharmaceutically acceptable carrier.E223. The method of embodiment 222, wherein the checkpoint inhibitorpolypeptide inhibits PD1, PD-L1, CTLA4, or a combination thereof.E224. The method of embodiment 223, wherein the checkpoint inhibitorpolypeptide is an antibody.E225. The method of embodiment 224, wherein the checkpoint inhibitorpolypeptide is an antibody selected from an anti-CTLA4 antibody orantigen-binding fragment thereof that specifically binds CTLA4, ananti-PD1 antibody or antigen-binding fragment thereof that specificallybinds PD1, an anti-PD-L1 antibody or antigen-binding fragment thereofthat specifically binds PD-L1, and a combination thereof.E226. The method of embodiment 225, wherein the checkpoint inhibitorpolypeptide is an anti-PD-L1 antibody selected from atezolizumab,avelumab, or durvalumab.E227. The method of embodiment 225, wherein the checkpoint inhibitorpolypeptide is an anti-CTLA-4 antibody selected from tremelimumab oripilimumab.E228. The method of embodiment 225, wherein the checkpoint inhibitorpolypeptide is an anti-PD1 antibody selected from nivolumab orpembrolizumab.E229. The method of any one of embodiments 222-228, wherein thecomposition comprising the checkpoint inhibitor polypeptide isadministered by intravenous injection.E230. The method of embodiment 229, wherein the composition comprisingthe checkpoint inhibitor polypeptide is administered once every 2 to 3weeks.E231. The method of embodiment 229, wherein the composition comprisingthe checkpoint inhibitor polypeptide is administered once every 2 weeksor once every 3 weeks.E232. The method of any one of embodiments 222-231, wherein thecomposition comprising the checkpoint inhibitor polypeptide isadministered prior to, concurrent with, or subsequent to administrationof the lipid nanoparticle or pharmaceutical composition thereof.E233. The method of any one of embodiments 217-232, wherein the subjecthas a histologically confirmed KRAS mutation selected from G12D, G12V,G13D or G12C.E234. The method of any one of embodiments 217-233, wherein the tumor ismetastatic colorectal cancer.E235. The method of any of embodiments 217-233, wherein the tumor isnon-small cell lung cancer (NSCLC).E236. The method of any of embodiments 217-233, wherein the tumor ispancreatic cancer.E237. An immunomodulatory therapeutic composition, comprising:one or more mRNA each having an open reading frame encoding anactivating oncogene mutation peptide, and a pharmaceutically acceptablecarrier or excipient.E238. The immunomodulatory therapeutic composition of embodiment 237,wherein the activating oncogene mutation is a KRAS mutationE239. The immunomodulatory therapeutic composition of embodiment 238,wherein the KRAS mutation is a G12 mutation.E240. The immunomodulatory therapeutic composition of embodiment 239,wherein the G12 KRAS mutation is selected from a G12D, G12V, G12S, G12C,G12A, and a G12R KRAS mutationE241. The immunomodulatory therapeutic composition of embodiment 239,wherein the G12 KRAS mutation is selected from a G12D, G12V, and a G12SKRAS mutation.E242. The immunomodulatory therapeutic composition of embodiment 238,wherein the KRAS mutation is a G13 mutation.E243. The immunomodulatory therapeutic composition of embodiment 242,wherein the G13 KRAS mutation is a G13D KRAS mutation.E244. The immunomodulatory therapeutic composition of embodiment 237,wherein the activating oncogene mutation is a H-RAS or N-RAS mutation.E245. The immunomodulatory therapeutic composition of any one ofembodiments 237-244, wherein the mRNA has an open reading frame encodinga concatemer of two or more activating oncogene mutation peptides.E246. The immunomodulatory therapeutic composition of embodiment 245,wherein at least two of the peptide epitopes are separated from oneanother by a single Glycine.E247. The immunomodulatory therapeutic composition of any one ofembodiments 245-246, wherein the concatemer comprises 3-10 activatingoncogene mutation peptides.E248. The activating oncogene mutation peptides of any one ofembodiments 245-247, wherein all of the peptide epitopes are separatedfrom one another by a single Glycine.E249. The activating oncogene mutation peptides of any one ofembodiments 245-247, wherein at least two of the peptide epitopes arelinked directly to one another without a linker.E250. The immunomodulatory therapeutic composition of any one ofembodiments 237-249, wherein the composition further comprises a cancertherapeutic agent.E251. The immunomodulatory therapeutic composition of any one ofembodiments 237-250, wherein the composition further comprises aninhibitory checkpoint polypeptide.E252. The immunomodulatory therapeutic composition of embodiment 251,wherein the inhibitory checkpoint polypeptide is an antibody or fragmentthereof that specifically binds to a molecule selected from the groupconsisting of PD-1, TIM-3, VISTA, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO,KIR and LAG3.E253. The immunomodulatory therapeutic composition of any one ofembodiments 237-252, wherein the composition further comprises a recallantigen.E254. The immunomodulatory therapeutic composition of embodiment 253,wherein the recall antigen is an infectious disease antigen.E255. The immunomodulatory therapeutic composition of any one ofembodiments 237-254, wherein the composition does not comprise astabilization agent.E256. The immunomodulatory therapeutic composition of any one ofembodiments 237-255, wherein the mRNA is formulated in a lipidnanoparticle carrier.E257. The immunomodulatory therapeutic composition of embodiment 256,wherein the lipid nanoparticle carrier comprises a molar ratio of about20-60% cationic lipid:5-25% non-cationic lipid:25-55% sterol; and0.5-15% PEG-modified lipid.E258. The immunomodulatory therapeutic composition of embodiment 257,wherein the cationic lipid is selected from the group consisting of forexample, 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane(DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate(DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319).E259. The immunomodulatory therapeutic composition of any one ofembodiments 237-258, wherein the mRNA includes at least one chemicalmodification.E260. The immunomodulatory therapeutic composition of embodiment 259,wherein the chemical modification is selected from the group consistingof pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4′-thiouridine,5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine,2-thio-dihydropseudouridine, 2-thio-dihydrouridine,2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine,4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine,4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine,5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2′-O-methyluridine.E261. A method for treating a subject, comprising: administering to asubject having cancer an immunomodulatory therapeutic composition of anyone of embodiments 237-260.E262. The method of embodiment 261, wherein immunomodulatory therapeuticcomposition isadministered in combination with a cancer therapeutic agent.E263. The method of embodiment 261 or 260, wherein immunomodulatorytherapeutic composition is administered in combination with aninhibitory checkpoint polypeptide.E264. The method of embodiment 263, wherein the inhibitory checkpointpolypeptide is an antibody or fragment thereof that specifically bindsto a molecule selected from the group consisting of PD-1, TIM-3, VISTA,A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR and LAG3.E265. The method of any one of embodiments 261-264, wherein the canceris selected from cancer of the pancreas, peritoneum, large intestine,small intestine, biliary tract, lung, endometrium, ovary, genital tract,gastrointestinal tract, cervix, stomach, urinary tract, colon, rectum,and hematopoietic and lymphoid tissues.E266. The method of embodiment 265, wherein the cancer is colorectalcancer.

Definitions

Administering: As used herein, “administering” refers to a method ofdelivering a composition to a subject or patient. A method ofadministration may be selected to target delivery (e.g., to specificallydeliver) to a specific region or system of a body. For example, anadministration may be parenteral (e.g., subcutaneous, intracutaneous,intravenous, intraperitoneal, intramuscular, intraarticular,intraarterial, intrasynovial, intrasternal, intrathecal, intralesional,or intracranial injection, as well as any suitable infusion technique),oral, trans- or intra-dermal, interdermal, rectal, intravaginal, topical(e.g. by powders, ointments, creams, gels, lotions, and/or drops),mucosal, nasal, buccal, enteral, vitreal, intratumoral, sublingual,intranasal; by intratracheal instillation, bronchial instillation,and/or inhalation; as an oral spray and/or powder, nasal spray, and/oraerosol, and/or through a portal vein catheter.

Approximately, about: As used herein, the terms “approximately” or“about,” as applied to one or more values of interest, refers to a valuethat is similar to a stated reference value. In certain embodiments, theterm “approximately” or “about” refers to a range of values that fallwithin 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%,8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greaterthan or less than) of the stated reference value unless otherwise statedor otherwise evident from the context (except where such number wouldexceed 100% of a possible value).

Cancer: As used herein, “cancer” is a condition involving abnormaland/or unregulated cell growth. The term cancer encompasses benign andmalignant cancers. Exemplary non-limiting cancers include adrenalcortical cancer, advanced cancer, anal cancer, aplastic anemia, bileductcancer, bladder cancer, bone cancer, bone metastasis, brain tumors,brain cancer, breast cancer, childhood cancer, cancer of unknown primaryorigin, Castleman disease, cervical cancer, colorectal cancer,endometrial cancer, esophagus cancer, Ewing family of tumors, eyecancer, gallbladder cancer, gastrointestinal carcinoid tumors,gastrointestinal stromal tumors, gestational trophoblastic disease,Hodgkin disease, Kaposi sarcoma, renal cell carcinoma, laryngeal andhypopharyngeal cancer, acute lymphocytic leukemia, acute myeloidleukemia, chronic lymphocytic leukemia, chronic myeloid leukemia,chronic myelomonocytic leukemia, myelodysplastic syndrome (includingrefractory anemias and refractory cytopenias), myeloproliferativeneoplasms or diseases (including polycythemia vera, essentialthrombocytosis and primary myelofibrosis), liver cancer (e.g.,hepatocellular carcinoma), non-small cell lung cancer, small cell lungcancer, lung carcinoid tumor, lymphoma of the skin, malignantmesothelioma, multiple myeloma, myelodysplasia syndrome, nasal cavityand paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma,non-Hodgkin lymphoma, oral cavity and oropharyngeal cancer,osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer,pituitary tumors, prostate cancer, retinoblastoma, rhabdomyosarcoma,salivary gland cancer, sarcoma in adult soft tissue, basal and squamouscell skin cancer, melanoma, small intestine cancer, stomach cancer,testicular cancer, throat cancer, thymus cancer, thyroid cancer, uterinesarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia,Wilms tumor and secondary cancers caused by cancer treatment. Inparticular embodiments, the cancer is liver cancer (e.g., hepatocellularcarcinoma) or colorectal cancer. In other embodiments, the cancer is ablood-based cancer or a hematopoetic cancer.

Cleavable Linker: As used herein, the term “cleavable linker” refers toa linker, typically a peptide linker (e.g., about 5-30 amino acids inlength, typically about 10-20 amino acids in length) that can beincorporated into multicistronic mRNA constructs such that equimolarlevels of multiple genes can be produced from the same mRNA.Non-limiting examples of cleavable linkers include the 2A family ofpeptides, including F2A, P2A, T2A and E2A, first discovered inpicornaviruses, that when incorporated into an mRNA construct (e.g.,between two polypeptide domains) function by making the ribosome skipthe synthesis of a peptide bond at C-terminus of the 2A element, therebyleading to separation between the end of the 2A sequence and the nextpeptide downstream.

Conjugated: As used herein, the term “conjugated,” when used withrespect to two or more moieties, means that the moieties are physicallyassociated or connected with one another, either directly or via one ormore additional moieties that serves as a linking agent, to form astructure that is sufficiently stable so that the moieties remainphysically associated under the conditions in which the structure isused, e.g., physiological conditions. In some embodiments, two or moremoieties may be conjugated by direct covalent chemical bonding. In otherembodiments, two or more moieties may be conjugated by ionic bonding orhydrogen bonding.

Contacting: As used herein, the term “contacting” means establishing aphysical connection between two or more entities. For example,contacting a cell with an mRNA or a lipid nanoparticle composition meansthat the cell and mRNA or lipid nanoparticle are made to share aphysical connection. Methods of contacting cells with external entitiesboth in vivo, in vitro, and ex vivo are well known in the biologicalarts. In exemplary embodiments of the disclosure, the step of contactinga mammalian cell with a composition (e.g., an isolated mRNA,nanoparticle, or pharmaceutical composition of the disclosure) isperformed in vivo. For example, contacting a lipid nanoparticlecomposition and a cell (for example, a mammalian cell) which may bedisposed within an organism (e.g., a mammal) may be performed by anysuitable administration route (e.g., parenteral administration to theorganism, including intravenous, intramuscular, intradermal, andsubcutaneous administration). For a cell present in vitro, a composition(e.g., a lipid nanoparticle or an isolated mRNA) and a cell may becontacted, for example, by adding the composition to the culture mediumof the cell and may involve or result in transfection. Moreover, morethan one cell may be contacted by a nanoparticle composition.

Encapsulate: As used herein, the term “encapsulate” means to enclose,surround, or encase. In some embodiments, a compound, polynucleotide(e.g., an mRNA), or other composition may be fully encapsulated,partially encapsulated, or substantially encapsulated. For example, insome embodiments, an mRNA of the disclosure may be encapsulated in alipid nanoparticle, e.g., a liposome.

Effective amount: As used herein, the term “effective amount” of anagent is that amount sufficient to effect beneficial or desired results,for example, clinical results, and, as such, an “effective amount”depends upon the context in which it is being applied. For example, inthe context of administering an agent that treats cancer, an effectiveamount of an agent is, for example, an amount sufficient to achievetreatment, as defined herein, of cancer, as compared to the responseobtained without administration of the agent. In some embodiments, atherapeutically effective amount is an amount of an agent to bedelivered (e.g., nucleic acid, drug, therapeutic agent, diagnosticagentor prophylactic agent) that is sufficient, when administered to asubject suffering from or susceptible to an infection, disease,disorder, and/or condition, to treat, improve symptoms of, diagnose,prevent, and/or delay the onset of the infection, disease, disorder,and/or condition.

Expression: As used herein, “expression” of a nucleic acid sequencerefers to one or more of the following events: (1) production of an RNAtemplate from a DNA sequence (e.g., by transcription); (2) processing ofan RNA transcript (e.g., by splicing, editing, 5′ cap formation, and/or3′ end processing); (3) translation of an RNA into a polypeptide orprotein; and (4) post-translational modification of a polypeptide orprotein.

Identity: As used herein, the term “identity” refers to the overallrelatedness between polymeric molecules, e.g., between polynucleotidemolecules (e.g., DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. Calculation of the percent identity of twopolynucleotide sequences, for example, can be performed by aligning thetwo sequences for optimal comparison purposes (e.g., gaps can beintroduced in one or both of a first and a second nucleic acid sequencesfor optimal alignment and non-identical sequences can be disregarded forcomparison purposes). In certain embodiments, the length of a sequencealigned for comparison purposes is at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, or 100% of the length of the reference sequence. The nucleotides atcorresponding nucleotide positions are then compared. When a position inthe first sequence is occupied by the same nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap which needs to be introduced for optimal alignment of the twosequences. The comparison of sequences and determination of percentidentity between two sequences can be accomplished using a mathematicalalgorithm. For example, the percent identity between two nucleotidesequences can be determined using methods such as those described inComputational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer,Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991;each of which is incorporated herein by reference. For example, thepercent identity between two nucleotide sequences can be determinedusing the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17), whichhas been incorporated into the ALIGN program (version 2.0) using aPAM120 weight residue table, a gap length penalty of 12 and a gappenalty of 4. The percent identity between two nucleotide sequences can,alternatively, be determined using the GAP program in the GCG softwarepackage using an NWSgapdna.CMP matrix. Methods commonly employed todetermine percent identity between sequences include, but are notlimited to those disclosed in Carillo, H., and Lipman, D., SIAM JApplied Math., 48:1073 (1988); incorporated herein by reference.Techniques for determining identity are codified in publicly availablecomputer programs. Exemplary computer software to determine homologybetween two sequences include, but are not limited to, GCG programpackage, Devereux et al., Nucleic Acids Research, 12(1): 387, 1984,BLASTP, BLASTN, and FASTA, Altschul, S. F. et al., J. Molec. Biol., 215,403, 1990.

Fragment: A “fragment,” as used herein, refers to a portion. Forexample, fragments of proteins may include polypeptides obtained bydigesting full-length protein isolated from cultured cells or obtainedthrough recombinant DNA techniques.

GC-rich: As used herein, the term “GC-rich” refers to the nucleobasecomposition of a polynucleotide (e.g., mRNA), or any portion thereof(e.g., an RNA element), comprising guanine (G) and/or cytosine (C)nucleobases, or derivatives or analogs thereof, wherein the GC-contentis greater than about 50%. The term “GC-rich” refers to all, or to aportion, of a polynucleotide, including, but not limited to, a gene, anon-coding region, a 5′ UTR, a 3′ UTR, an open reading frame, an RNAelement, a sequence motif, or any discrete sequence, fragment, orsegment thereof which comprises about 50% GC-content. In someembodiments of the disclosure, GC-rich polynucleotides, or any portionsthereof, are exclusively comprised of guanine (G) and/or cytosine (C)nucleobases.

GC-content: As used herein, the term “GC-content” refers to thepercentage of nucleobases in a polynucleotide (e.g., mRNA), or a portionthereof (e.g., an RNA element), that are either guanine (G) and cytosine(C) nucleobases, or derivatives or analogs thereof, (from a total numberof possible nucleobases, including adenine (A) and thymine (T) or uracil(U), and derivatives or analogs thereof, in DNA and in RNA). The term“GC-content” refers to all, or to a portion, of a polynucleotide,including, but not limited to, a gene, a non-coding region, a 5′ or 3′UTR, an open reading frame, an RNA element, a sequence motif, or anydiscrete sequence, fragment, or segment thereof.

Genetic Adjuvant: A “genetic adjuvant”, as used herein, refers to anmRNA construct (e.g., an mmRNA construct) that enhances the immuneresponse to a vaccine, for example by stimulating cytokine productionand/or by stimulating the production of antigen-specific effector cells(e.g., CD8 T cells). A genetic adjuvant mRNA construct can, for example,encode a polypeptide that stimulates Type I interferon (e.g., activatesType I interferon pathway signaling) or that promotes dendritic celldevelopment or activity.

Heterologous: As used herein, “heterologous” indicates that a sequence(e.g., an amino acid sequence or the polynucleotide that encodes anamino acid sequence) is not normally present in a given polypeptide orpolynucleotide. For example, an amino acid sequence that corresponds toa domain or motif of one protein may be heterologous to a secondprotein.

Hydrophobic amino acid: As used herein, a “hydrophobic amino acid” is anamino acid having an uncharged, nonpolar side chain. Examples ofnaturally occurring hydrophobic amino acids are alanine (Ala), valine(Val), leucine (Leu), isoleucine (Ile), proline (Pro), phenylalanine(Phe), methionine (Met), and tryptophan (Trp).

Immune Potentiator: An “immune potentiator”, as used herein, refers toan mRNA construct (e.g., an mmRNA construct) that enhances an immuneresponse, e.g., to an antigen of interest (either an endogenous antigenin a subject to which the immune potentiator is administered or to anexogenous antigen that is coadministered with the immune potentiator),for example by stimulating T cell, B cell or dendritic cell responses,including but not limited to cytokine production, stimulating antibodyproduction or stimulating the production of antigen-specific immunecells (e.g., CD8⁺ T cells or CD4⁺ T cells).

Initiation Codon: As used herein, the term “initiation codon”, usedinterchangeably with the term “start codon”, refers to the first codonof an open reading frame that is translated by the ribosome and iscomprised of a triplet of linked adenine-uracil-guanine nucleobases. Theinitiation codon is depicted by the first letter codes of adenine (A),uracil (U), and guanine (G) and is often written simply as “AUG”.Although natural mRNAs may use codons other than AUG as the initiationcodon, which are referred to herein as “alternative initiation codons”,the initiation codons of polynucleotides described herein use the AUGcodon. During the process of translation initiation, the sequencecomprising the initiation codon is recognized via complementarybase-pairing to the anticodon of an initiator tRNA (Met-tRNA_(i) ^(Met))bound by the ribosome. Open reading frames may contain more than one AUGinitiation codon, which are referred to herein as “alternate initiationcodons”.

The initiation codon plays a critical role in translation initiation.The initiation codon is the first codon of an open reading frame that istranslated by the ribosome. Typically, the initiation codon comprisesthe nucleotide triplet AUG, however, in some instances translationinitiation can occur at other codons comprised of distinct nucleotides.The initiation of translation in eukaryotes is a multistep biochemicalprocess that involves numerous protein-protein, protein-RNA, and RNA-RNAinteractions between messenger RNA molecules (mRNAs), the 40S ribosomalsubunit, other components of the translation machinery (e.g., eukaryoticinitiation factors; eIFs). The current model of mRNA translationinitiation postulates that the pre-initiation complex (alternatively“43S pre-initiation complex”; abbreviated as “PIC”) translocates fromthe site of recruitment on the mRNA (typically the 5′ cap) to theinitiation codon by scanning nucleotides in a 5′ to 3′ direction untilthe first AUG codon that resides within a specific translation-promotivenucleotide context (the Kozak sequence) is encountered (Kozak (1989) JCell Biol 108:229-241). Scanning by the PIC ends upon complementarybase-pairing between nucleotides comprising the anticodon of theinitiator Met-tRNA_(i) ^(Met) transfer RNA and nucleotides comprisingthe initiation codon of the mRNA. Productive base-pairing between theAUG codon and the Met-tRNA_(i) ^(Met) anticodon elicits a series ofstructural and biochemical events that culminate in the joining of thelarge 60S ribosomal subunit to the PIC to form an active ribosome thatis competent for translation elongation.

Insertion: As used herein, an “insertion” or an “addition” refers to achange in an amino acid or nucleotide sequence resulting in the additionof one or more amino acid residues or nucleotides, respectively, to amolecule as compared to a reference sequence, for example, the sequencefound in a naturally-occurring molecule. In some embodiments, aninsertion may be a replacement.

Insertion Site: As used herein, an “insertion site” is a position orregion of a scaffold polypeptide that is amenable to insertion of anamino acid sequence of a heterologous polypeptide. It is to beunderstood that an insertion site also may refer to the position orregion of the polynucleotide that encodes the polypeptide (e.g., a codonof a polynucleotide that codes for a given amino acid in the scaffoldpolypeptide). In some embodiments, insertion of an amino acid sequenceof a heterologous polypeptide into a scaffold polypeptide has little tono effect on the stability (e.g., conformational stability), expressionlevel, or overall secondary structure of the scaffold polypeptide.

Isolated: As used herein, the term “isolated” refers to a substance orentity that has been separated from at least some of the components withwhich it was associated (whether in nature or in an experimentalsetting). Isolated substances may have varying levels of purity inreference to the substances from which they have been associated.Isolated substances and/or entities may be separated from at least about10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%,about 80%, about 90%, or more of the other components with which theywere initially associated. In some embodiments, isolated agents are morethan about 80%, about 85%, about 90%, about 91%, about 92%, about 93%,about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, ormore than about 99% pure. As used herein, a substance is “pure” if it issubstantially free of other components.

Kozak Sequence: The term “Kozak sequence” (also referred to as “Kozakconsensus sequence”) refers to a translation initiation enhancer elementto enhance expression of a gene or open reading frame, and which ineukaryotes, is located in the 5′ UTR. The Kozak consensus sequence wasoriginally defined as the sequence GCCRCC, where R=a purine, followingan analysis of the effects of single mutations surrounding theinitiation codon (AUG) on translation of the preproinsulin gene (Kozak(1986) Cell 44:283-292). Polynucleotides disclosed herein comprise aKozak consensus sequence, or a derivative or modification thereof.(Examples of translational enhancer compositions and methods of usethereof, see U.S. Pat. No. 5,807,707 to Andrews et al., incorporatedherein by reference in its entirety; U.S. Pat. No. 5,723,332 toChernajovsky, incorporated herein by reference in its entirety; U.S.Pat. No. 5,891,665 to Wilson, incorporated herein by reference in itsentirety.) Leaky scanning: A phenomenon known as “leaky scanning” canoccur whereby the PIC bypasses the initiation codon and insteadcontinues scanning downstream until an alternate or alternativeinitiation codon is recognized. Depending on the frequency ofoccurrence, the bypass of the initiation codon by the PIC can result ina decrease in translation efficiency. Furthermore, translation from thisdownstream AUG codon can occur, which will result in the production ofan undesired, aberrant translation product that may not be capable ofeliciting the desired therapeutic response. In some cases, the aberranttranslation product may in fact cause a deleterious response (Kracht etal., (2017) Nat Med 23(4):501-507).

Liposome: As used herein, by “liposome” is meant a structure including alipid-containing membrane enclosing an aqueous interior. Liposomes mayhave one or more lipid membranes. Liposomes include single-layeredliposomes (also known in the art as unilamellar liposomes) andmulti-layered liposomes (also known in the art as multilamellarliposomes).

Metastasis: As used herein, the term “metastasis” means the process bywhich cancer spreads from the place at which it first arose as a primarytumor to distant locations in the body. A secondary tumor that arose asa result of this process may be referred to as “a metastasis.”

Modified: As used herein “modified” or “modification” refers to achanged state or a change in composition or structure of apolynucleotide (e.g., mRNA). Polynucleotides may be modified in variousways including chemically, structurally, and/or functionally. Forexample, polynucleotides may be structurally modified by theincorporation of one or more RNA elements, wherein the RNA elementcomprises a sequence and/or an RNA secondary structure(s) that providesone or more functions (e.g., translational regulatory activity).Accordingly, polynucleotides of the disclosure may be comprised of oneor more modifications (e.g., may include one or more chemical,structural, or functional modifications, including any combinationthereof).

mRNA: As used herein, an “mRNA” refers to a messenger ribonucleic acid.An mRNA may be naturally or non-naturally occurring. For example, anmRNA may include modified and/or non-naturally occurring components suchas one or more nucleobases, nucleosides, nucleotides, or linkers. AnmRNA may include a cap structure, a chain terminating nucleoside, a stemloop, a polyA sequence, and/or a polyadenylation signal. An mRNA mayhave a nucleotide sequence encoding a polypeptide. Translation of anmRNA, for example, in vivo translation of an mRNA inside a mammaliancell, may produce a polypeptide. Traditionally, the basic components ofan mRNA molecule include at least a coding region, a 5′-untranslatedregion (5′-UTR), a 3′UTR, a 5′ cap and a polyA sequence.

microRNA (miRNA): As used herein, a “microRNA (miRNA)” is a smallnon-coding RNA molecule which may function in post-transcriptionalregulation of gene expression (e.g., by RNA silencing, such as bycleavage of the mRNA, destabilization of the mRNA by shortening itspolyA tail, and/or by interfering with the efficiency of translation ofthe mRNA into a polypeptide by a ribosome). A mature miRNA is typicallyabout 22 nucleotides long.

microRNA-122 (miR-122): As used herein, “microRNA-122 (miR-122)” refersto any native miR-122 from any vertebrate source, including, forexample, humans, unless otherwise indicated. miR-122 is typically highlyexpressed in the liver, where it may regulate fatty-acid metabolism.miR-122 levels are reduced in liver cancer, for example, hepatocellularcarcinoma. miR-122 is one of the most highly-expressed miRNAs in theliver, where it regulates targets including but not limited to CAT-1,CD320, AldoA, Hjv, Hfe, ADAM10, IGFR1, CCNG1, and ADAM17. Mature humanmiR-122 may have a sequence of AACGCCAUUAUCACACUAAAUA (SEQ ID NO: 172,corresponding to hsa-miR-122-3p) or UGGAGUGUGACAAUGGUGUUUG (SEQ ID NO:174, corresponding to hsa-miR-122-5p).

microRNA-21 (miR-21): As used herein, “microRNA-21 (miR-21)” refers toany native miR-21 from any vertebrate source, including, for example,humans, unless otherwise indicated. miR-21 levels are increased in livercancer, for example, hepatocellular carcinoma, as compared to normalliver. Mature human miR-21 may have a sequence of UAGCUUAUCAGACUGAUGUUGA(SEQ ID NO: 34, corresponding to has-miR-21-5p) or5′-CAACACCAGUCGAUGGGCUGU-3′ (SEQ ID NO: 35, corresponding tohas-miR-21-3p).

microRNA-142 (miR-142): As used herein, “microRNA-142 (miR-142)” refersto any native miR-142 from any vertebrate source, including, forexample, humans, unless otherwise indicated. miR-142 is typically highlyexpressed in myeloid cells. Mature human miR-142 may have a sequence ofUGUAGUGUUUCCUACUUUAUGGA (SEQ ID NO: 28, corresponding to hsa-miR-142-3p)or CAUAAAGUAGAAAGCACUACU (SEQ ID NO: 30, corresponding tohsa-miR-142-5p).

microRNA (miRNA) binding site: As used herein, a “microRNA (miRNA)binding site” refers to a miRNA target site or a miRNA recognition site,or any nucleotide sequence to which a miRNA binds or associates. In someembodiments, a miRNA binding site represents a nucleotide location orregion of a polynucleotide (e.g., an mRNA) to which at least the “seed”region of a miRNA binds. It should be understood that “binding” mayfollow traditional Watson-Crick hybridization rules or may reflect anystable association of the miRNA with the target sequence at or adjacentto the microRNA site.

miRNA seed: As used herein, a “seed” region of a miRNA refers to asequence in the region of positions 2-8 of a mature miRNA, whichtypically has perfect Watson-Crick complementarity to the miRNA bindingsite. A miRNA seed may include positions 2-8 or 2-7 of a mature miRNA.In some embodiments, a miRNA seed may comprise 7 nucleotides (e.g.,nucleotides 2-8 of a mature miRNA), wherein the seed-complementary sitein the corresponding miRNA binding site is flanked by an adenine (A)opposed to miRNA position 1. In some embodiments, a miRNA seed maycomprise 6 nucleotides (e.g., nucleotides 2-7 of a mature miRNA),wherein the seed-complementary site in the corresponding miRNA bindingsite is flanked by an adenine (A) opposed to miRNA position 1. Whenreferring to a miRNA binding site, an miRNA seed sequence is to beunderstood as having complementarity (e.g., partial, substantial, orcomplete complementarity) with the seed sequence of the miRNA that bindsto the miRNA binding site.

Modified: As used herein “modified” refers to a changed state orstructure of a molecule of the disclosure. Molecules may be modified inmany ways including chemically, structurally, and functionally. In oneembodiment, the mRNA molecules of the present disclosure are modified bythe introduction of non-natural nucleosides and/or nucleotides, e.g., asit relates to the natural ribonucleotides A, U, G, and C. Noncanonicalnucleotides such as the cap structures are not considered “modified”although they differ from the chemical structure of the A, C, G, Uribonucleotides.

Nanoparticle: As used herein, “nanoparticle” refers to a particle havingany one structural feature on a scale of less than about 1000 nm thatexhibits novel properties as compared to a bulk sample of the samematerial. Routinely, nanoparticles have any one structural feature on ascale of less than about 500 nm, less than about 200 nm, or about 100nm. Also routinely, nanoparticles have any one structural feature on ascale of from about 50 nm to about 500 nm, from about 50 nm to about 200nm or from about 70 to about 120 mn. In exemplary embodiments, ananoparticle is a particle having one or more dimensions of the order ofabout 1-1000 nm. In other exemplary embodiments, a nanoparticle is aparticle having one or more dimensions of the order of about 10-500 nm.In other exemplary embodiments, a nanoparticle is a particle having oneor more dimensions of the order of about 50-200 nm. A sphericalnanoparticle would have a diameter, for example, of between about 50-100or 70-120 nanometers. A nanoparticle most often behaves as a unit interms of its transport and properties. it is noted that novel propertiesthat differentiate nanoparticles from the corresponding bulk materialtypically develop at a size scale of under 1000 nm, or at a size ofabout 100 nm, but nanoparticles can be of a larger size, for example,for particles that are oblong, tubular, and the like. Although the sizeof most molecules would fit into the above outline, individual moleculesare usually not referred to as nanoparticles.

Nucleic acid: As used herein, the term “nucleic acid” is used in itsbroadest sense and encompasses any compound and/or substance thatincludes a polymer of nucleotides. These polymers are often referred toas polynucleotides. Exemplary nucleic acids or polynucleotides of thedisclosure include, but are not limited to, ribonucleic acids (RNAs),deoxyribonucleic acids (DNAs), DNA-RNA hybrids, RNAi-inducing agents,RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes,catalytic DNA, RNAs that induce triple helix formation, threose nucleicacids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs),locked nucleic acids (LNAs, including LNA having a β-D-riboconfiguration, a-LNA having an a-L-ribo configuration (a diastereomer ofLNA), 2′-amino-LNA having a 2′-amino functionalization, and2′-amino-a-LNA having a 2′-amino functionalization) or hybrids thereof.

Nucleic Acid Structure: As used herein, the term “nucleic acidstructure” (used interchangeably with “polynucleotide structure”) refersto the arrangement or organization of atoms, chemical constituents,elements, motifs, and/or sequence of linked nucleotides, or derivativesor analogs thereof, that comprise a nucleic acid (e.g., an mRNA). Theterm also refers to the two-dimensional or three-dimensional state of anucleic acid. Accordingly, the term “RNA structure” refers to thearrangement or organization of atoms, chemical constituents, elements,motifs, and/or sequence of linked nucleotides, or derivatives or analogsthereof, comprising an RNA molecule (e.g., an mRNA) and/or refers to atwo-dimensional and/or three dimensional state of an RNA molecule.Nucleic acid structure can be further demarcated into fourorganizational categories referred to herein as “molecular structure”,“primary structure”, “secondary structure”, and “tertiary structure”based on increasing organizational complexity.

Nucleobase: As used herein, the term “nucleobase” (alternatively“nucleotide base” or “nitrogenous base”) refers to a purine orpyrimidine heterocyclic compound found in nucleic acids, including anyderivatives or analogs of the naturally occurring purines andpyrimidines that confer improved properties (e.g., binding affinity,nuclease resistance, chemical stability) to a nucleic acid or a portionor segment thereof. Adenine, cytosine, guanine, thymine, and uracil arethe nucleobases predominately found in natural nucleic acids. Othernatural, non-natural, and/or synthetic nucleobases, as known in the artand/or described herein, can be incorporated into nucleic acids.

Nucleoside/Nucleotide: As used herein, the term “nucleoside” refers to acompound containing a sugar molecule (e.g., a ribose in RNA or adeoxyribose in DNA), or derivative or analog thereof, covalently linkedto a nucleobase (e.g., a purine or pyrimidine), or a derivative oranalog thereof (also referred to herein as “nucleobase”), but lacking aninternucleoside linking group (e.g., a phosphate group). As used herein,the term “nucleotide” refers to a nucleoside covalently bonded to aninternucleoside linking group (e.g., a phosphate group), or anyderivative, analog, or modification thereof that confers improvedchemical and/or functional properties (e.g., binding affinity, nucleaseresistance, chemical stability) to a nucleic acid or a portion orsegment thereof.

Open Reading Frame: As used herein, the term “open reading frame”,abbreviated as “ORF”, refers to a segment or region of an mRNA moleculethat encodes a polypeptide. The ORF comprises a continuous stretch ofnon-overlapping, in-frame codons, beginning with the initiation codonand ending with a stop codon, and is translated by the ribosome.

Patient: As used herein, “patient” refers to a subject who may seek orbe in need of treatment, requires treatment, is receiving treatment,will receive treatment, or a subject who is under care by a trainedprofessional for a particular disease or condition. In particularembodiments, a patient is a human patient. In some embodiments, apatient is a patient suffering from cancer (e.g., liver cancer orcolorectal cancer).

Pharmaceutically acceptable: The phrase “pharmaceutically acceptable” isemployed herein to refer to those compounds, materials, compositions,and/or dosage forms which are, within the scope of sound medicaljudgment, suitable for use in contact with the tissues of human beingsand animals without excessive toxicity, irritation, allergic response,or other problem or complication, commensurate with a reasonablebenefit/risk ratio

Pharmaceutically acceptable excipient: The phrase “pharmaceuticallyacceptable excipient,” as used herein, refers any ingredient other thanthe compounds described herein (for example, a vehicle capable ofsuspending or dissolving the active compound) and having the propertiesof being substantially nontoxic and non-inflammatory in a patient.Excipients may include, for example: antiadherents, antioxidants,binders, coatings, compression aids, disintegrants, dyes (colors),emollients, emulsifiers, fillers (diluents), film formers or coatings,flavors, fragrances, glidants (flow enhancers), lubricants,preservatives, printing inks, sorbents, suspensing or dispersing agents,sweeteners, and waters of hydration. Exemplary excipients include, butare not limited to: butylated hydroxytoluene (BHT), calcium carbonate,calcium phosphate (dibasic), calcium stearate, croscarmellose,crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine,ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol,methionine, methylcellulose, methyl paraben, microcrystalline cellulose,polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinizedstarch, propyl paraben, retinyl palmitate, shellac, silicon dioxide,sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate,sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide,vitamin A, vitamin E, vitamin C, and xylitol.

Pharmaceutically acceptable salts: As used herein, “pharmaceuticallyacceptable salts” refers to derivatives of the disclosed compoundswherein the parent compound is modified by converting an existing acidor base moiety to its salt form (e.g., by reacting the free base groupwith a suitable organic acid). Examples of pharmaceutically acceptablesalts include, but are not limited to, mineral or organic acid salts ofbasic residues such as amines; alkali or organic salts of acidicresidues such as carboxylic acids; and the like. Representative acidaddition salts include acetate, acetic acid, adipate, alginate,ascorbate, aspartate, benzenesulfonate, benzene sulfonic acid, benzoate,bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate,hexanoate, hydrobromide, hydrochloride, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, toluenesulfonate, undecanoate, valerate salts, and thelike. Representative alkali or alkaline earth metal salts includesodium, lithium, potassium, calcium, magnesium, and the like, as well asnontoxic ammonium, quaternary ammonium, and amine cations, including,but not limited to ammonium, tetramethylammonium, tetraethylammonium,methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine,and the like. The pharmaceutically acceptable salts of the presentdisclosure include the conventional non-toxic salts of the parentcompound formed, for example, from non-toxic inorganic or organic acids.The pharmaceutically acceptable salts of the present disclosure can besynthesized from the parent compound which contains a basic or acidicmoiety by conventional chemical methods. Generally, such salts can beprepared by reacting the free acid or base forms of these compounds witha stoichiometric amount of the appropriate base or acid in water or inan organic solvent, or in a mixture of the two; generally, nonaqueousmedia like ether, ethyl acetate, ethanol, isopropanol, or acetonitrileare preferred. Lists of suitable salts are found in Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa.,1985, p. 1418, Pharmaceutical Salts: Properties, Selection, and Use, P.H. Stahl and C. G. Wermuth (eds.), Wiley-VCH, 2008, and Berge et al.,Journal of Pharmaceutical Science, 66, 1-19 (1977), each of which isincorporated herein by reference in its entirety.

Polypeptide: As used herein, the term “polypeptide” or “polypeptide ofinterest” refers to a polymer of amino acid residues typically joined bypeptide bonds that can be produced naturally (e.g., isolated orpurified) or synthetically.

Pre-Initiation Complex (PIC): As used herein, the term “pre-initiationcomplex” (alternatively “43S pre-initiation complex”; abbreviated as“PIC”) refers to a ribonucleoprotein complex comprising a 40S ribosomalsubunit, eukaryotic initiation factors (eIF1, eIF1A, eIF3, eIF5), andthe eIF2-GTP-Met-tRNA_(i) ^(M)et ternary complex, that is intrinsicallycapable of attachment to the 5′ cap of an mRNA molecule and, afterattachment, of performing ribosome scanning of the 5′ UTR.

RNA element: As used herein, the term “RNA element” refers to a portion,fragment, or segment of an RNA molecule that provides a biologicalfunction and/or has biological activity (e.g., translational regulatoryactivity). Modification of a polynucleotide by the incorporation of oneor more RNA elements, such as those described herein, provides one ormore desirable functional properties to the modified polynucleotide. RNAelements, as described herein, can be naturally-occurring, non-naturallyoccurring, synthetic, engineered, or any combination thereof. Forexample, naturally-occurring RNA elements that provide a regulatoryactivity include elements found throughout the transcriptomes ofviruses, prokaryotic and eukaryotic organisms (e.g., humans). RNAelements in particular eukaryotic mRNAs and translated viral RNAs havebeen shown to be involved in mediating many functions in cells.Exemplary natural RNA elements include, but are not limited to,translation initiation elements (e.g., internal ribosome entry site(IRES), see Kieft et al., (2001) RNA 7(2):194-206), translation enhancerelements (e.g., the APP mRNA translation enhancer element, see Rogers etal., (1999) J Biol Chem 274(10):6421-6431), mRNA stability elements(e.g., AU-rich elements (AREs), see Garneau et al., (2007) Nat Rev MolCell Biol 8(2):113-126), translational repression element (see e.g.,Blumer et al., (2002) Mech Dev 110(1-2):97-112), protein-binding RNAelements (e.g., iron-responsive element, see Selezneva et al., (2013) JMol Biol 425(18):3301-3310), cytoplasmic polyadenylation elements(Villalba et al., (2011) Curr Opin Genet Dev 21(4):452-457), andcatalytic RNA elements (e.g., ribozymes, see Scott et al., (2009)Biochim Biophys Acta 1789(9-10):634-641).

Residence time: As used herein, the term “residence time” refers to thetime of occupancy of a pre-initiation complex (PIC) or a ribosome at adiscrete position or location along an mRNA molecule.

Substantially: As used herein, the term “substantially” refers to thequalitative condition of exhibiting total or near-total extent or degreeof a characteristic or property of interest. One of ordinary skill inthe biological arts will understand that biological and chemicalphenomena rarely, if ever, go to completion and/or proceed tocompleteness or achieve or avoid an absolute result. The term“substantially” is therefore used herein to capture the potential lackof completeness inherent in many biological and chemical phenomena.

Suffering from: An individual who is “suffering from” a disease,disorder, and/or condition has been diagnosed with or displays one ormore symptoms of a disease, disorder, and/or condition.

Targeting moiety: As used herein, a “targeting moiety” is a compound oragent that may target a nanoparticle to a particular cell, tissue,and/or organ type.

Therapeutic Agent: The term “therapeutic agent” refers to any agentthat, when administered to a subject, has a therapeutic, diagnostic,and/or prophylactic effect and/or elicits a desired biological and/orpharmacological effect.

Transfection: As used herein, the term “transfection” refers to methodsto introduce a species (e.g., a polynucleotide, such as a mRNA) into acell.

Translational Regulatory Activity: As used herein, the term“translational regulatory activity” (used interchangeably with“translational regulatory function”) refers to a biological function,mechanism, or process that modulates (e.g., regulates, influences,controls, varies) the activity of the translational apparatus, includingthe activity of the PIC and/or ribosome. In some aspects, the desiredtranslation regulatory activity promotes and/or enhances thetranslational fidelity of mRNA translation. In some aspects, the desiredtranslational regulatory activity reduces and/or inhibits leakyscanning. Subject: As used herein, the term “subject” refers to anyorganism to which a composition in accordance with the disclosure may beadministered, e.g., for experimental, diagnostic, prophylactic, and/ortherapeutic purposes. Typical subjects include animals (e.g., mammalssuch as mice, rats, rabbits, non-human primates, and humans) and/orplants. In some embodiments, a subject may be a patient.

Treating: As used herein, the term “treating” refers to partially orcompletely alleviating, ameliorating, improving, relieving, delayingonset of, inhibiting progression of, reducing severity of, and/orreducing incidence of one or more symptoms or features of a particularinfection, disease, disorder, and/or condition. For example, “treating”cancer may refer to inhibiting survival, growth, and/or spread of atumor. Treatment may be administered to a subject who does not exhibitsigns of a disease, disorder, and/or condition and/or to a subject whoexhibits only early signs of a disease, disorder, and/or condition forthe purpose of decreasing the risk of developing pathology associatedwith the disease, disorder, and/or condition.

Preventing: As used herein, the term “preventing” refers to partially orcompletely inhibiting the onset of one or more symptoms or features of aparticular infection, disease, disorder, and/or condition.

Tumor: As used herein, a “tumor” is an abnormal growth of tissue,whether benign or malignant.

Unmodified: As used herein, “unmodified” refers to any substance,compound or molecule prior to being changed in any way. Unmodified may,but does not always, refer to the wild type or native form of abiomolecule. Molecules may undergo a series of modifications wherebyeach modified molecule may serve as the “unmodified” starting moleculefor a subsequent modification.

Uridine Content: The terms “uridine content” or “uracil content” areinterchangeable and refer to the amount of uracil or uridine present ina certain nucleic acid sequence. Uridine content or uracil content canbe expressed as an absolute value (total number of uridine or uracil inthe sequence) or relative (uridine or uracil percentage respect to thetotal number of nucleobases in the nucleic acid sequence).

Uridine-Modified Sequence: The terms “uridine-modified sequence” refersto a sequence optimized nucleic acid (e.g., a synthetic mRNA sequence)with a different overall or local uridine content (higher or loweruridine content) or with different uridine patterns (e.g., gradientdistribution or clustering) with respect to the uridine content and/oruridine patterns of a candidate nucleic acid sequence. In the content ofthe present disclosure, the terms “uridine-modified sequence” and“uracil-modified sequence” are considered equivalent andinterchangeable.

A “high uridine codon” is defined as a codon comprising two or threeuridines, a “low uridine codon” is defined as a codon comprising oneuridine, and a “no uridine codon” is a codon without any uridines. Insome embodiments, a uridine-modified sequence comprises substitutions ofhigh uridine codons with low uridine codons, substitutions of highuridine codons with no uridine codons, substitutions of low uridinecodons with high uridine codons, substitutions of low uridine codonswith no uridine codons, substitution of no uridine codons with lowuridine codons, substitutions of no uridine codons with high uridinecodons, and combinations thereof. In some embodiments, a high uridinecodon can be replaced with another high uridine codon. In someembodiments, a low uridine codon can be replaced with another lowuridine codon. In some embodiments, a no uridine codon can be replacedwith another no uridine codon. A uridine-modified sequence can beuridine enriched or uridine rarefied.

Uridine Enriched: As used herein, the terms “uridine enriched” andgrammatical variants refer to the increase in uridine content (expressedin absolute value or as a percentage value) in a sequence optimizednucleic acid (e.g., a synthetic mRNA sequence) with respect to theuridine content of the corresponding candidate nucleic acid sequence.Uridine enrichment can be implemented by substituting codons in thecandidate nucleic acid sequence with synonymous codons containing lessuridine nucleobases. Uridine enrichment can be global (i.e., relative tothe entire length of a candidate nucleic acid sequence) or local (i.e.,relative to a subsequence or region of a candidate nucleic acidsequence).

Uridine Rarefied: As used herein, the terms “uridine rarefied” andgrammatical variants refer to a decrease in uridine content (expressedin absolute value or as a percentage value) in an sequence optimizednucleic acid (e.g., a synthetic mRNA sequence) with respect to theuridine content of the corresponding candidate nucleic acid sequence.Uridine rarefication can be implemented by substituting codons in thecandidate nucleic acid sequence with synonymous codons containing lessuridine nucleobases. Uridine rarefication can be global (i.e., relativeto the entire length of a candidate nucleic acid sequence) or local(i.e., relative to a subsequence or region of a candidate nucleic acidsequence).

EQUIVALENTS AND SCOPE

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments in accordance with the disclosure described herein. Thescope of the present disclosure is not intended to be limited to theDescription below, but rather is as set forth in the appended claims.

In the claims, articles such as “a,” “an,” and “the” may mean one ormore than one unless indicated to the contrary or otherwise evident fromthe context. Claims or descriptions that include “or” between one ormore members of a group are considered satisfied if one, more than one,or all of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The disclosure includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Thedisclosure includes embodiments in which more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process.

It is also noted that the terms “comprising”, “comprise”, “comprises”,“having”, “have” and “has” are intended to be open and permit but doesnot require the inclusion of additional elements or steps. When theseterms are used herein, the term “consisting of” is thus also encompassedand disclosed.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and understanding of one of ordinary skill in the art, valuesthat are expressed as ranges can assume any specific value or subrangewithin the stated ranges in different embodiments of the disclosure, tothe tenth of the unit of the lower limit of the range, unless thecontext clearly dictates otherwise.

All cited sources, for example, references, publications, databases,database entries, and art cited herein, are incorporated into thisapplication by reference, even if not expressly stated in the citation.In case of conflicting statements of a cited source and the instantapplication, the statement in the instant application shall control.

EXAMPLES

The disclosure will be more fully understood by reference to thefollowing examples. They should not, however, be construed as limitingthe scope of the disclosure. It is understood that the examples andembodiments described herein are for illustrative purposes only and thatvarious modifications or changes in light thereof will be suggested topersons skilled in the art and are to be included within the spirit andpurview of this application and scope of the appended claims.

Example 1: STING Immune Potentiator mRNA Constructs

In this example, a series of mmRNA constructs that encodedconstitutively activated forms of human STING were made and tested fortheir ability to stimulate interferon-β (IFN-β) production. The humanSTING protein encoded by the constructs was constitutively activatedthrough introduction of one or more point mutations. The followingsingle or combination point mutations were tested: (i) V155M; (ii)R284T; (iii) V147L/N154S/V155M; and (iv) R284M/V147L/N154S/V155M. Theseconstructs typically also encoded an epitope tag at either theN-terminus or C-terminus to facilitate detection. Different epitope tagswere tested (FLAG, Myc, CT, HA, V5). Additionally, all constructscontained a Cap 1 5′ Cap (7mG(5′)ppp(5′)NlmpNp), 5′ UTR, 3′ UTR, a polyA tail of 100 nucleotides and were fully modified with1-methyl-pseudouridine (m1ψ). The ORF amino acid sequences ofrepresentative constitutively active human STING constructs without anyepitope tag are shown in SEQ ID NOs: 1-10. An exemplary 5′ UTR for usein the constructs is shown in SEQ ID NO: 21. An exemplary 3′ UTR for usein the constructs is shown in SEQ ID NO: 22. An exemplary 3′ UTRcomprising miR-122 and miR-142.3p binding sites for use in theconstructs is shown in SEQ ID NO: 23.

To determine whether constitutively active STING constructs couldstimulate IFN-β production, the constructs were transfected into humanTF1a cells. Wild-type (non-constitutively active) human and mouse STINGconstructs were used as negative controls. Twenty-five thousandcells/well were plated in 96 well plates and the mmRNA constructs (250ng) were transfected into them using Lipofectamine 2000. After 24 and 48hours, supernatants were harvested and IFN-β levels were determined bystandard ELISA. The results are shown in FIG. 1, which demonstrate thatthe constitutively active STING constructs stimulated IFN-β production,as compared to the wild-type (non-constitutively active) human and mouseSTING controls. While all four mutant STING constructs stimulated IFN-βproduction, the V155M mutant and the R284T mutant showed the highestactivity. These results demonstrate the ability of constitutively activeSTING mRNA constructs to enhance immune responses through stimulation ofIFN-β production.

In a second set of experiments, a reporter gene whose transcription wasdriven by an interferon-sensitive response element (ISRE) was used totest the ability of a panel of constitutively active STING mRNAconstructs to activate the ISRE in a STING KO reporter mouse line. Theresults are shown in FIG. 2, which demonstrates that the constitutivelyactive STING constructs stimulated reporter gene expression, therebyindicating that the constructs were capable of activating theinterferon-sensitive response element (ISRE).

Example 2: IRF3 and IRF7 Immune Potentiator mRNA Constructs

In this example, a series of mmRNA constructs that encodedconstitutively activated forms of IRF3 or IRF7 were made and tested fortheir ability to activate an interferon-sensitive response element(ISRE). The ORF amino acid sequences of representative constitutivelyactive mouse and human IRF3 constructs, comprising a S396D pointmutation, without any epitope tag are shown in SEQ ID NOs: 11-12. TheORF amino acid sequence of a wild-type human IRF7 construct without anyepitope tag is shown in SEQ ID NO: 13. The ORF amino acid sequences ofrepresentative constitutively active human IRF7 constructs without anyepitope tag are shown in SEQ ID NOs: 14-18. The ORF amino acid sequencesof representative truncated human IRF7 constructs (inactive “null”mutations) without any epitope tag are shown in SEQ ID NOs: 19-20. Theseconstructs typically also encoded an epitope tag at either theN-terminus or C-terminus to facilitate detection. Different epitope tagswere tested (FLAG, Myc, CT, HA, V5). Additionally, all constructscontained a Cap 1 5′ Cap (7mG(5′)ppp(5′)NlmpNp), 5′ UTR, 3′ UTR, a polyA tail of 100 nucleotides and were fully modified with1-methyl-pseudouridine (m1ψ). An exemplary 5′ UTR for use in theconstructs is shown in SEQ ID NO: 21. An exemplary 3′ UTR for use in theconstructs is shown in SEQ ID NO: 22. An exemplary 3′ UTR comprisingmiR-122 and miR-142.3p binding sites for use in the constructs is shownin SEQ ID NO: 23.

A reporter gene whose transcription was driven by aninterferon-sensitive response element (ISRE) was used to test theability of constitutively active IRF3 and IRF7 mRNA constructs toactivate the ISRE. The results are shown in FIGS. 3A-3B, whichdemonstrate that the constitutively active IRF3 constructs (FIG. 3A) andthe constitutively active IRF7 constructs (FIG. 3B) stimulated reportergene expression, thereby indicating that the constructs were capable ofactivating the interferon-sensitive response element (ISRE).

Example 3: IKKβ, cFLIP and RIPK1 Immune Potentiator mRNA Constructs

In this example, a luciferase reporter gene whose transcription wasdriven by the NFκB signaling pathway was used to test the ability ofconstitutively active IKK, cFLIP and RIPK1 mRNA constructs to activateNFκB signaling.

Constitutively active IKKβ construct comprised the following two pointmutations: S177E/S181E. Constitutively active IKKα or IKKβ constructscomprised PEST mutations. The ORF amino acid sequences of constitutivelyactive IKKβ constructs without any epitope tag are shown in SEQ ID NOs:87-90. The ORF amino acid sequences of constitutively active IKKα orIKKβ constructs comprising a PEST mutation, without any epitope tag, areshown in SEQ ID NOs: 91-98. Constitutively active cFLIP constructscomprised cFLIP-L, cFLIP-S (aa 1-227), cFLIP p22 (aa 1-198), cFLIP p43(aa 1-376) or cFLIP p12 (aa 377-480). The ORF amino acid sequences ofthe cFLIP constructs without any epitope tag are shown in SEQ ID NOs:82-86. Structures of various constitutively active RIPK1 constructs aredescribed further in, for example, Yatim, N. et al. (2015) Science350:328-334 or Orozco, S. et al. (2014) Cell Death Differ. 21:1511-1521.The ORF amino acid sequences of the constitutively active RIPK1constructs without any epitope tag are shown in SEQ ID NOs: 99-104. Inaddition to the open reading frame, all constructs contained a Cap 1 5′Cap (7mG(5′)ppp(5′)NlmpNp), 5′ UTR, 3′ UTR, a poly A tail of 100nucleotides and were fully modified with 1-methyl-pseudouridine (m1ψ).An exemplary 5′ UTR for use in the constructs is shown in SEQ ID NO: 21.An exemplary 3′ UTR for use in the constructs is shown in SEQ ID NO: 22.An exemplary 3′ UTR comprising miR-122 and miR-142.3p binding sites foruse in the constructs is shown in SEQ ID NO: 23.

In a first series of experiments, either the cFLIP or IKKβ constructs(12.5 ng RNA) were transfected into B16F10, MC38 or HEK293 cells,together with the NFκB-luc reporter gene and the Dual Luc Assay wasperformed 24 hours post-transfection as an indicator of activation ofNFκB signaling. The results are shown in FIG. 4, which demonstrates thatthe constitutively active cFLIP and IKKβ constructs stimulated reportergene expression, thereby indicating that the constructs were capable ofactivating the NFκB signaling pathway. In a second series ofexperiments, the RIPK1 constructs were transfected into B16F10 cells,together with the NFκB-luc reporter gene and the Dual Luc Assay wasperformed 24 hours post-transfection as an indicator of activation ofNFκB signaling. The results are shown in FIG. 5, which demonstrates thatthe constitutively active RIPK1 constructs stimulated reporter geneexpression, thereby indicating that the constructs were capable ofactivating the NFκB signaling pathway.

Example 4: DIABLO Immune Potentiator mRNA Constructs

In this example, a series of mmRNA constructs that encoded DIABLO weremade and tested for their ability to induce cytokine production. Theseconstructs typically also encoded an epitope tag at either theN-terminus or C-terminus to facilitate detection. Different epitope tagswere tested (FLAG, Myc, CT, HA, V5). Additionally, all constructscontained a Cap 1 5′ Cap (7mG(5′)ppp(5′)NlmpNp), 5′ UTR, 3′ UTR, a polyA tail of 100 nucleotides and were fully modified with1-methyl-pseudouridine (m1ψ). The ORF amino acid sequences of the DIABLOconstructs without any epitope tag are shown in SEQ ID NOs: 106-113. Anexemplary 5′ UTR for use in the constructs is shown in SEQ ID NO: 21. Anexemplary 3′ UTR for use in the constructs is shown in SEQ ID NO: 22. Anexemplary 3′ UTR comprising miR-122 and miR-142.3p binding sites for usein the constructs is shown in SEQ ID NO: 23.

To determine wither the DIABLO constructs could induce cytokineproduction, the constructs were transfected into SKOV3 cells. Tenthousand cells/well were plated in 96 well plates and the mmRNAconstructs were transfected into them using Lipofectamine 2000.Stimulation of cytokine production by the DIABLO mmRNA constructs in theSKOV3 cells was measured. The results, shown in FIG. 6 for TNF-α and inFIG. 7 for interleukin 6 (IL-6), demonstrate that a number of the DIABLOmmRNA constructs stimulate production of cytokines by the SKOV3 cells.

Example 5: Immune Potentiator mRNAs Enhance MC38 Cancer VaccineResponses

In this example, the potency and durability of responses to an MC38mRNA-based cancer vaccine used in combination with STING, IRF3 or IRF7immune potentiator mRNA constructs were examined. The MC38 murine tumormodel has been used to identify immunogenic mutant peptides containingneoepitopes capable of stimulating anti-tumor T cell responses (seee.g., Yadav, M. et al. (2014) Nature 515:572-576). Thus, a cancervaccination approach that leads to a robust and durable immune responseagainst tumor neoepitopes is highly desirable.

The MC38 vaccine used in this example was an mRNA construct encoding anADR concatemer of three 25mer mutant peptides containing tumorneoepitopes derived from Adpgk, Dpagt1, and Reps1 (this vaccine is alsoreferred to herein as ADRvax). The mRNA construct encodes the openreading frame shown in SEQ ID NO: 120, which also includes an N-terminalHis-tag for easy detection. Mice were immunized intramuscularly with theADRvax mRNA vaccine (at a dose of 0.25 mg/kg) on days 0 and 14,combination with either a control mRNA construct (NTFIX), or a STING,IRF3 or IRF7 immune potentiator mRNA construct (at a dose of 0.25mg/kg). The constitutively active STING immune potentiator contained aV155M mutation (mouse version corresponding to SEQ ID NO: 1). Theconstitutively active IRF3 immune potentiator contained a S396D mutation(corresponding to SEQ ID NO: 12). The constitutively active IRF7 immunepotentiator contained an internal deletion and six point mutations(mouse version corresponding to SEQ ID NO: 18). The MC38 vaccineconstruct and the genetic adjuvant construct were coformulated in MC3lipid nanoparticles.

At day 21 and 35, CD8⁺ spleen cells from mice in each test group wererestimulated ex vivo for 4 hours at 37 degrees C. in the presence ofGolgiPlug™ (containing Brefeldin A; BD Biosciences) with eitherwild-type or mutant MC38 ADR peptides (1 μg/ml per peptide) and CD8vaccine responses were assessed by intracellular staining (ICS) forIFN-γ. Representative ICS results for MC38 ADR-specific responses by day21 and day 35 CD8⁺spleen cells for IFN-γ are shown in FIG. 8A (day 21)and FIG. 8B (day 31). Similar results were observed for ICS for TNF-αand for CD8⁺PBMCs. The results demonstrate that CD8 vaccine responseswere greatly enhanced by the STING immune potentiator construct, andmoderately enhanced by the IRF3 and IRF7 immune potentiator constructs.An initial improvement in the antigen-specific CD8 response for micetreated with immune potentiators was observed at day 21 (approximately5% versus 1% for STING treatment vs. control), which continued toimprove by day 35 (up to 15% for STING treatment compared to control),thereby demonstrating the durability of the response.

The percentage of CD8b⁺ cells among the live CD45⁺ cells was alsoexamined. The results for day 35 spleen cells and PBMCs are shown inFIG. 9A, which demonstrates that the genetic adjuvants expand the totalCD8b⁺ population. As demonstrated in FIG. 9B, the majority of the CD8⁺spleen cells and PBMCs were found to have an “effector memory”CD62L^(lo) phenotype. Additional staining experiments demonstrated thatthe STING and IRF7 immune potentiator construct slightly reduced the %of total Foxp3⁺ Treg CD4 Tcells (data not shown). Additional stainingexperiments demonstrated that the immune potentiators did not change the% of CD138⁺ plasmablasts (data not shown).

Example 6: KRAS-STING mRNA Constructs

A comprehensive survey of Ras mutations in various cancer types has beenreported (Prior, I. A. et al. (2012) Cancer Res. 72:2457-2467). Thissurvey demonstrated that the top four most frequent mutations of KRAS incolorectal cancer, pancreatic cancer and non-small cell lung cancer areG12D, G12V, G13D and G12C. A series of mutant KRAS mRNA constructs wereprepared that encoded one or more KRAS peptides containing one of thesefour mutations, for use as KRAS anti-tumor mRNA-based vaccines.Furthermore, to examine the effect of mRNA-based immune potentiators onKRAS vaccine responses, a series of mRNA constructs were prepared thatencoded one or more mutant KRAS peptides linked at the N-terminus or theC-terminus to sequence encoding STING as an immune potentiator. Thus, inthese KRAS-STING mRNA constructs, the vaccine antigen(s) and the immunepotentiator are encoded by the same mRNA construct.

Mutant KRAS peptide mRNA constructs were prepared that encoded: a 15merpeptide having the G12D, G12V or the G13D mutation (the amino acidsequence of which is shown in SEQ ID NOs: 36-38, respectively); a 25merpeptide having the G12D, G12V or the G13D mutation (SEQ ID NOs: 39-41,respectively); three copies of the 15mer peptide having the G12D, G12Vor the G13D mutation (SEQ ID NOs: 42-44, respectively); or three copiesof the 25mer peptide having the G12D, G12V or the G13D mutation (SEQ IDNOs: 45-47, respectively). Additional constructs encoded one copy orthree copies of a 25mer peptide having a G12C mutation (SEQ ID NOs:72-73, respectively) or a wild-type 25mer peptide (SEQ ID NO: 74). Incertain embodiments, a G12C KRAS mutation may be used in combinationwith a G12D, G12V or G13D mutation, or combinations thereof. Nucleotidesequences encoding these mutant KRAS peptides are provided in Example 7.

Mutant KRAS peptide-STING mRNA constructs, having the STING codingsequence at the N-terminus, were prepared that encoded: a 15mer peptidehaving the G12D, G12V or the G13D mutation (the amino acid sequence ofwhich is shown in SEQ ID NOs: 48-50, respectively); a 25mer peptidehaving the G12D, G12V or the G13D mutation (SEQ ID NOs: 51-53,respectively); three copies of the 15mer peptide having the G12D, G12Vor the G13D mutation (SEQ ID NOs: 54-56, respectively); or three copiesof the 25mer peptide having the G12D, G12V or the G13D mutation (SEQ IDNOs: 57-59, respectively). In certain embodiments, a G12C KRAS mutationmay be used in combination with a G12D, G12V or G13D mutation, orcombinations thereof. Representative nucleotide sequences encoding theseKRAS peptide-STING constructs having the STING coding sequence at theN-terminus are shown in SEQ ID NOs: 160 and 162.

Mutant KRAS peptide-STING mRNA constructs, having the STING codingsequence at the C-terminus, were prepared that encoded: a 15mer peptidehaving the G12D, G12V or the G13D mutation (the amino acid sequence ofwhich is shown in SEQ ID NOs: 60-62, respectively); a 25mer peptidehaving the G12D, G12V or the G13D mutation (SEQ ID NOs: 63-65,respectively); three copies of the 15mer peptide having the G12D, G12Vor the G13D mutation (SEQ ID NOs: 66-68, respectively); or three copiesof the 25mer peptide having the G12D, G12V or the G13D mutation (SEQ IDNOs: 69-70, respectively). In certain embodiments, a G12C KRAS mutationmay be used in combination with a G12D, G12V or G13D mutation, orcombinations thereof. Representative nucleotide sequences encoding theseKRAS peptide-STING constructs having the STING coding sequence at theC-terminus are shown in SEQ ID NOs: 161 and 163.

These constructs can also encoded an epitope tag at either theN-terminus or C-terminus to facilitate detection. Different epitope tagscan be used (e.g., FLAG, Myc, CT, HA, V5). Additionally, all constructscontained a Cap 1 5′ Cap (7mG(5′)ppp(5′)NlmpNp), 5′ UTR, 3′ UTR, a polyA tail and were fully modified with 1-methyl-pseudouridine (m1ψ). Anexemplary 5′ UTR for use in the constructs is shown in SEQ ID NO: 21. Anexemplary 3′ UTR for use in the constructs is shown in SEQ ID NO: 22. Anexemplary 3′ UTR comprising miR-122 and miR-142.3p binding sites for usein the constructs is shown in SEQ ID NO: 23.

To test vaccine responses in mice treated either with a KRAS mutantpeptide(s) mRNA vaccine construct or with a KRAS mutant peptide(s)vaccine-STING immune potentiator mRNA construct, mice (HLA-A*11:01 orHLA-A*2:01; Taconic) are immunized with a KRAS mutant peptide vaccinemRNA construct (e.g., encoding one of SEQ ID NOs: 36-47) or with a KRASmutant peptide vaccine-STING immune potentiator mRNA construct (e.g.,encoding one of SEQ ID NOs: 48-71). Mice are immunized intramuscularlyon day 1 and day 15 (0.5 mg/kg) and sacrificed at day 22. To test CD8vaccine responses, CD8⁺ spleen cells and PBMCs are restimulated ex vivofor 5 hours at 37 degrees C. in the presence of GolgiPlug™ (containingBrefeldin A; BD Biosciences) with either mutant KRAS peptides (G12D,G12V or G13D) or with wild type KRAS peptide (2 μg/ml per peptide). CD8vaccine responses can then be assessed by intracellular staining (ICS)for IFN-γ and/or TNF-α. Enhanced ICS responses for IFN-γ and/or TNF-α inmice treated with the KRAS mutant peptide vaccine-STING immunepotentiator mRNA construct, as compared to treatment with the KRASmutant peptide vaccine mRNA construct, indicates that the STING immunepotentiator enhances KRAS-specific CD8 vaccine responses.

Example 7: Use of Immune Potentiator mRNA Construct in Combination withActivating Oncogene KRAS Mutant Peptide mRNA Constructs

In this example, mutant KRAS peptide mRNA constructs are used incombination with a separate constitutively active STING immunepotentiator mRNA construct to enhance immune responses to the mutantKRAS peptides.

The most frequently mutated oncogene in cancer is KRAS, which is mutatedin roughly 30% of epithelial cancers, primarily lung, colorectal andpancreatic cancers (Pylayeva-Gupta Y, et al., Nat Rev Cancer, Vol.11(11): 761-774, 2011). The 4 most prevalent KRAS mutant antigens inthese three malignancies are G12D, G12V, G13D and G12C, which constitute80-90% of the KRAS mutations (Prior et al. Cancer Res. 2012 May 15;72(10): 2457-2467; Cox A D et al, Nat Rev Drug Discov, Vol. 13(11):828-851, 2011). KRAS mutations occur mostly in a couple of “hotspots”and activate the oncogene. Prior research has shown limited ability toraise T cells specific to the oncogenic mutation. However, much of thiswas done in the context of the most common HLA allele (A2, which occursin ˜50% of Caucasians). More recently, it has been demonstrated that (a)specific T cells can be generated against point mutations in the contextof less common HLA alleles (A11, C8), and (b) growing these cellsex-vivo and transferring them back to the patient has mediated adramatic tumor response in a patient with lung cancer. (N Engl J Med2016; 375:2255-2262 Dec. 8, 2016 DOI: 10.1056/NEJMoa1609279).

KRAS mutations occur in approximately 40% of colorectal cancers. Asshown in Table 5 below, in CRC (colorectal cancer), only 3 mutations(G12V, G12D, and G13D) account for 96% of KRAS mutations in thismalignancy. Furthermore, all CRC patients get typed for KRAS mutationsas standard of care.

TABLE 5 COSMIC* case counts All cancers % CRC % G12S 1849 1% G12V 92134% 5215 29% G12C 435 2% G12D 13634 7% 8083 44% G12A 2179 1% G12R 1244 1%G13D 5084 2% 4267 23% 18% 96% Tested 208629 18271*http://cancer.sanger.ac.uk/cosmic/gene/analysis?In=KRAS

In another COSMIC data set, 73.68% of KRAS mutations in colorectalcancer are accounted for by these 3 mutations (G12V, G12D, and G13D)(Table 6).

TABLE 6 colon % rectal % total % 12D 635 35.04 178 33.46 813 34.68 12V364 20.09 124 23.31 488 20.82 13D 338 18.65 88 16.54 426 18.17 73.68

Prior et al. investigated and summarized isoform-specific point mutationspecificity for HRAS, KRAS and NRAS, respectively. Data representingtotal number of tumors with each point mutation were collated fromCOSMIC v52 release. The most frequent mutations for each isoform foreach cancer type are reported (see Table 2 of Prior et al.). Inaddition, secondary KRAS mutations have been identified in EGFR blockaderesistant patients. RAS is downstream of EGFR and it has been found toconstitute a mechanism of resistance to EGFR blockade therapies. EGFRblockade resistant KRAS mutant tumors can be targeted using compositionsand methods disclosed herein. In a few cases, more than one KRASmutation was identified in the same patient (up to four differentmutations co-occur). Diaz et al. report these secondary KRAS mutationsafter acquisition of EGFR blockade (see Supplementary Table 2), andMisale et al. reports secondary KRAS mutations after EGFR blockade (seeFIG. 3b ) (Diaz et al. The molecular evolution of acquired resistance totargeted EGFR blockade in colorectal cancers, Nature 486: 537 (2012);Misale et al. Emergence of KRAS mutations and acquired resistance toanti-EGFR therapy in colorectal cancer, Nature 486: 532 (2012)). Thismutational spectrum appears to be at least somewhat different thanprimary tumor missense mutants in colorectal cancer. As shown in FIG.10, NRAS is also mutated in colorectal cancer, but at a lower frequencythan KRAS, based on analysis available in cBioPortal and Prior et al.

In addition to identification of KRAS mutations in colorectal cancer,such mutations have been found in non-small cell lung carcinoma andpancreatic cancer. Table 7 provides the frequencies of four KRASmutations in these three cancers.

TABLE 7 NSCLC¹ (30% mutant Colorectal² Pancreatic³ KRAS KRAS⁴) (45%mutant KRAS⁴) (95% mutant KRAS⁴) Allele % Breakdown % Breakdown %Breakdown G12C 46% 8% 2% G12V 20% 22% 30% G12D 11% 36% 51% G13D 3% 19%<1% total 80% 85% 83% ¹Mellema et al. Comparison of clinical outcomeafter first-line platinum-based chemotherapy in different types of KRASmutated advanced NSCLC, Lung Cancer 90: 2 (2015) (Table 1) ²Neumann etal, Frequency and type of KRAS mutations in routine diagnostic analysisof metastatic colorectal cancer, Pathology Research and Practice 205(2009) (FIG. 1) ³Kirsten L. Bryant, Joseph D. Mancias, Alec C.Kimmelman, Channing J. Der, KRAS: feeding pancreatic cancerproliferation, In Trends in Biochemical Sciences, 39: 2, 2014 (FIG. 2)⁴Adrienne D. Cox et al., Drugging the undruggable RAS: MissionPossible?, Nature Reviews Drug Discovery 13, 828-851 (2014) (Table 1)

In this example, animals are administered an immunomodulatorytherapeutic composition that includes an mRNA encoding at least oneactivating oncogene mutation peptide, e.g., at least one activating KRASmutation, alone or in combination with an immune potentiator mRNAconstruct, e.g. a constitutively active STING mRNA construct, e.g.,encoding a sequence as shown in any of SEQ ID NOs: 1-10, such as forexample a mRNA construct encoding a constitutively active human STINGprotein comprising a V155M mutation, having the amino acid sequenceshown in SEQ ID NO: 1 and encoded the nucleotide sequence shown in SEQID NO: 139.

Exemplary KRAS mutant peptide sequences and mRNA constructs are shown inTables 8-10.

TABLE 8 KRAS mutant peptide sequences 9 AA sequence 15mer 25mer G12DVVGADGVGK MKLVVVGADGVGKSAL MTEYKLVVVGADGVGKSALTIQLIQ (SEQ ID NO: 121)(SEQ ID NO: 36) (SEQ ID NO: 39) G12V VVGAVGVGK MKLVVVGAVGVGKSALMTEYKLVVVGAVGVGKSALTIQLIQ (SEQ ID NO: 122) (SEQ ID NO: 37) (SEQ ID NO:40) G13D VGAGDVGKS MLVVVGAGDVGKSALT MTEYKLVVVGAGDVGKSALTIQLIQ (SEQ IDNO: 123) (SEQ ID NO: 38) (SEQ ID NO: 41) G12C VVGACGVGK MKLVVVGACGVGKSAMTEYKLVVVGACGVGKSALTIQLIQ (SEQ ID NO: 124) (SEQ ID NO: 125) (SEQ ID NO:72) WT MTEYKLVVVGAGGVGKSALTIQLIQ (SEQ ID NO: 74)

TABLE 9 KRAS mutant amino acid sequences KRAS MUTANT AMINO ACID SEQUENCEKRAS(G12D)15mer MKLVVVGADGVGKSAL (SEQ ID NO: 36) KRAS(G12V)15merMKLVVVGAVGVGKSAL (SEQ ID NO: 37) KRAS(G13D)15mer MLVVVGAGDVGKSALT (SEQID NO: 38) KRAS(G12D)25mer MTEYKLVVVGADGVGKSALTIQLIQ (SEQ ID NO: 39)KRAS(G12V)25mer MTEYKLVVVGAVGVGKSALTIQLIQ (SEQ ID NO: 40)KRAS(G13D)25mer MTEYKLVVVGAGDVGKSALTIQLIQ (SEQ ID NO: 41)KRAS(G12D)15mer^3 MKLVVVGADGVGKSALKLVVVGADGVGKSALKLVVVGADGVG KSAL (SEQID NO: 42) KRAS(G12V)15mer^3 MKLVVVGAVGVGKSALKLVVVGAVGVGKSALKLVVVGAVGVGKSAL (SEQ ID NO: 43) KRAS(G13D)15mer^3MLVVVGAGDVGKSALTLVVVGAGDVGKSALTLVVVGAGDVGK SALT (SEQ ID NO: 44)KRAS(G12D)25mer^3 MTEYKLVVVGADGVGKSALTIQLIQMTEYKLVVVGADGVGKSALTIQLIQMTEYKLVVVGADGVGKSALTIQLIQ (SEQ ID NO: 45) KRAS(G12V)25mer^3MTEYKLVVVGAVGVGKSALTIQLIQMTEYKLVVVGAVGVGKSALTIQLIQMTEYKLVVVGAVGVGKSALTIQLIQ (SEQ ID NO: 46) KRAS(G13D)25mer^3MTEYKLVVVGAGDVGKSALTIQLIQMTEYKLVVVGAGDVGKSALTIQLIQMTEYKLVVVGAGDVGKSALTIQLIQ (SEQ ID NO: 47) KRAS(G12C)25merMTEYKLVVVGACGVGKSALTIQLIQ (SEQ ID NO: 72) KRAS(G12C)25mer^3MTEYKLVVVGACGVGKSALTIQLIQMTEYKLVVVGACGVGKSALTIQLIQMTEYKLVVVGACGVGKSALTIQLIQ (SEQ ID NO: 73) KRAS(WT)25merMTEYKLVVVGAGGVGKSALTIQLIQ (SEQ ID NO: 74)

TABLE 10 KRAS mutant antigen mRNA sequences mRNA Orf Sequence (AminoName Acid) Orf Sequence (Nucleotide) KRAS MTEYKLVVVGADGATGACCGAGTACAAGCTGGTGGTGGTGGGCGCCG (G12D) VGKSALTIQLIQACGGCGTGGGCAAGAGCGCCCTGACCATCCAGCT 25mer (SEQ ID NO: 39) GATCCAG (SEQ IDNO: 126) KRAS MTEYKLVVVGAVG ATGACCGAGTACAAGCTGGTGGTGGTGGGCGCCG (G12V)VGKSALTIQLIQ TGGGCGTGGGCAAGAGCGCCCTGACCATCCAGCT 25mer (SEQ ID NO: 40)GATCCAG (SEQ ID NO: 127) KRAS MTEYKLVVVGAGDATGACCGAGTACAAGCTGGTGGTGGTGGGCGCCG (G13D) VGKSALTIQLIQGCGACGTGGGCAAGAGCGCCCTGACCATCCAGCT 25mer (SEQ ID NO: 41) GATCCAG (SEQ IDNO: 128) KRAS MTEYKLVVVGADG ATGACCGAGTACAAGTTAGTGGTTGTGGGCGCCG (G12D)VGKSALTIQLIQMTE ACGGCGTGGGCAAGAGCGCCCTCACCATCCAGCT 25mer^3 YKLVVVGADGVGKTATCCAGATGACGGAATATAAGTTAGTAGTAGTG SALTIQLIQMTEYKLGGAGCCGACGGTGTCGGCAAGTCCGCTTTGACCA VVVGADGVGKSALTTCAACTTATTCAGATGACAGAGTATAAGCTGGTC TIQLIQ (SEQ ID NO:GTTGTAGGCGCAGACGGCGTTGGAAAGTCGGCAC 45) TGACGATCCAGTTGATCCAG (SEQ ID NO:129) KRAS MTEYKLVVVGAVG ATGACCGAGTACAAGCTCGTCGTGGTGGGCGCCG (G12V)VGKSALTIQLIQMTE TGGGCGTGGGCAAGAGCGCCCTAACCATCCAGTT 25mer^3 YKLVVVGAVGVGKGATCCAGATGACCGAATATAAGCTCGTGGTAGTC SALTIQLIQMTEYKLGGAGCGGTGGGCGTTGGCAAGTCAGCGCTAACAA VVVGAVGVGKSALTACAACTAATCCAAATGACCGAATACAAGCTAGT TIQLIQ (SEQ ID NO:TGTAGTCGGTGCCGTCGGCGTTGGAAAGTCAGCC 46) CTTACAATTCAGCTCATTCAG (SEQ ID NO:130) KRAS MTEYKLVVVGAGD ATGACCGAGTACAAGCTCGTAGTGGTTGGCGCCG (G13D)VGKSALTIQLIQMTE GCGACGTGGGCAAGAGCGCCCTAACCATCCAGCT 25mer^3 YKLVVVGAGDVGKCATCCAGATGACAGAATATAAGCTTGTGGTTGTG SALTIQLIQMTEYKLGGAGCAGGAGACGTGGGAAAGAGTGCGTTGACG VVVGAGDVGKSALATTCAACTCATACAGATGACCGAATACAAGTTGG TIQLIQ (SEQ ID NO:TGGTGGTCGGCGCAGGTGACGTTGGTAAGTCTGC 47) ACTAACTATACAACTGATCCAG (SEQ IDNO: 190) KRAS MTEYKLVVVGACG ATGACCGAGTACAAGCTGGTGGTGGTGGGCGCCT (G12C)VGKSALTIQLIQ GCGGCGTGGGCAAGAGCGCCCTGACCATCCAGCT 25mer (SEQ ID NO: 72)GATCCAG (SEQ ID NO: 132) KRAS MTEYKLVVVGACGATGACCGAGTACAAGCTCGTGGTTGTTGGCGCCTG (G12C) VGKSALTIQLIQMTECGGCGTGGGCAAGAGCGCCCTCACCATCCAGCTC 25mer^3 YKLVVVGACGVGKATCCAGATGACAGAGTATAAGTTAGTCGTTGTCG SALTIQLIQMTEYKLGAGCTTGCGGAGTTGGAAAGTCGGCGCTCACCAT VVVGACGVGKSALTCAACTCATACAAATGACAGAATATAAGTTAGTG TIQLIQ (SEQ ID NO:GTGGTGGGTGCGTGTGGCGTTGGCAAGAGTGCGC 73) TTACTATCCAGCTCATTCAG (SEQ ID NO:184) KRAS MTEYKLVVVGAGG ATGACCGAGTACAAGCTGGTGGTGGTGGGCGCCG (WT)VGKSALTIQLIQ GCGGCGTGGGCAAGAGCGCCCTGACCATCCAGCT 25mer (SEQ ID NO: 74)GATCCAG (SEQ ID NO: 133) Chemistry: uridines modified N1-methylpseudouridine (m1Ψ) Cap: C1 Tail: T100 5′ UTR Sequence (standard5′ Flank (includes Production FP + T7 site + 5′UTR)):TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACC (SEQ ID NO: 21) 5′ UTR Sequence (NoPromoter): GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACC (SEQ ID NO:134) 3′ UTR Sequence (Human 3′ UTR no XbaI):TGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC (SEQ ID NO:22)

In a first study to examine the effect of a STING immune potentiatormRNA construct on KRAS antigen responses in vivo, HLA-A*2:01 Tg mice(Taconic, strain 9659F, n=4) are administered mRNA encoding variousforms of mutated KRAS peptide antigens as follows: mRNA encoding mutatedKRAS (alone or in combination with STING) administered on day 1, bleedtaken on day 8, mRNA encoding mutated KRAS (alone or in combination withSTING) administered on day 15, animal sacrificed on day 22. The testgroups are shown in Table 11 as follows:

TABLE 11 Test/Control Immune Dosing TEST group Group MaterialPotentiator Vehicle Route Regimen KRAS-MUT 1 KRAS G12D None (NTFIX)Compound 25 IM Day 1, 15 2 KRAS G12V None (NTFIX) Compound 25 IM Day 1,15 3 KRAS G13D None (NTFIX) Compound 25 IM Day 1, 15 4 KRAS G12C None(NTFIX) Compound 25 IM Day 1, 15 KRAS-MUT + 5 KRAS G12D STING (V155M)Compound 25 IM Day 1, 15 STING 6 KRAS G12V STING (V155M) Compound 25 IMDay 1, 15 7 KRAS G13D STING (V155M) Compound 25 IM Day 1, 15 8 KRAS G12CSTING (V155M) Compound 25 IM Day 1, 15 No Ag 9 NTFIX NTFIX Compound 25IM Day 1, 15 STING Only 10 NTFIX STING V155M) Compound 25 IM Day 1, 15

mRNA is administered to animals at a dose of 0.5 mg/kg (10 ug per 20-ganimal). The KRAS and STING constructs are administered at a 1:1 ratio.Ex vivo restimulation (2 ug/ml per peptide) is tested for 4 hours at 37degrees Celsius in the presence of GolgiPlug (Brefeldin A).Intracellular cytokine staining (ICS) is tested for KRAS G12D, KRASG12V, KRAS G13D, KRAS WT, and no peptide.

mRNA encoding KRAS mutations, alone or in combination with mRNA encodingconstitutively active STING, is tested for the ability to generate Tcells. Efficacy of mRNA encoding KRAS mutations is compared, forexample, to peptide vaccination. The effect of the STING immunepotentiator is determined by comparing treatment with the KRAS mutantpeptides alone versus in combination with the STING immune potentiator.For example, CD8 vaccine responses can be assessed by intracellularstaining (ICS) for IFN-γ and/or TNF-α as described herein. Enhanced ICSresponses for IFN-γ and/or TNF-α in mice treated with the KRAS mutantpeptide vaccine in combination with the STING immune potentiator mRNAconstruct, as compared to treatment with the KRAS mutant peptide vaccinemRNA construct alone, indicates that the STING immune potentiatorenhances KRAS-specific CD8 vaccine responses.

In a second study to examine the effect of the STING immune potentiatormRNA construct on immune responses to various different forms of themutant KRAS peptide antigen mRNA constructs, HLA*A*11:01 Tg mice(Taconic, strain 9660F, n=4) are administered mRNA encoding variousdifferent forms of mutated KRAS peptide antigen mRNA constructs incombination with a STING immune potentiator mRNA construct as follows:mRNA encoding mutated KRAS in combination with STING administered on day1, bleed taken on day 8, mRNA encoding mutated KRAS in combination withSTING administered on day 15, animal sacrificed on day 22.

The types of mutated KRAS constructs tested were as follows: (i) mRNAencoding a single mutant KRAS 25mer peptide antigen containing eitherthe G12D, G12V, G13D or G12C mutation (“singlet”); (ii) mRNA encoding aconcatemer of three 25mer peptide antigens (thus creating a 75mer), oneof each containing the G12D, G12V and G13D mutations (“KRAS-3MUT”);(iii) mRNA encoding a concatemer of four 25mer peptide antigens (thuscreating a 100mer), one of each containing the G12D, G12V, G13D and G12Cmutations (“KRAS-4MUT”); or (iv) four separate mRNAs coadministeredtogether, each encoding a single mutant KRAS 25mer peptide antigencontaining either the G12D, G12V, G13D or G12C mutation (“Single×4”).

The amino acid and nucleotide sequences of the G12D 25mer are shown inSEQ ID NOs: 39 and 126, respectively. The amino acid and nucleotidesequences of the G12V 25mer are shown in SEQ ID NOs: 40 and 127,respectively. The amino acid and nucleotide sequences of the G13D 25merare shown in SEQ ID NOs: 41 and 128, respectively. The amino acid andnucleotide sequences of the G12C 25mer are shown in SEQ ID NOs: 72 and132 respectively. The amino acid and nucleotide sequences of theKRAS-3MUT 75mer are shown in SEQ ID NOs: 135 and 136, respectively. Theamino acid and nucleotide sequences of the KRAS-4MUT 100mer are shown inSEQ ID NOs: 137 and 138, respectively.

The test groups are shown in Table 12 as follows:

TABLE 12 Test/Control Immune Dosing TEST group Group MaterialPotentiator Vehicle Route Regimen KRAS-MUT 1 KRAS G12D STING (V155M)Compound 25 IM Day 1, 15 Singlet 2 KRAS G12V STING (V155M) Compound 25IM Day 1, 15 3 KRAS G13D STING (V155M) Compound 25 IM Day 1, 15 4 KRASG12C STING (V155M) Compound 25 IM Day 1, 15 KRAS-MUT 5 KRAS-3MUT STING(V155M) Compound 25 IM Day 1, 15 Concatemer 6 KRAS-4MUT STING (V155M)Compound 25 IM Day 1, 15 Single X 4 7 G12D + G12V + STING (V155M)Compound 25 IM Day 1, 15 G12C + G13D STING Only 8 NTFIX STING (V155M)Compound 25 IM Day 1, 15

mRNA is administered to animals at a dose of 0.5 mg/kg (10 ug per 20-ganimal). The KRAS and STING constructs are administered at a 5:1 ratio.Ex vivo restimulation (2 ug/ml per peptide) is carried out for 5 hoursat 37 degrees Celsius in the presence of GolgiPlug (Brefeldin A).Intracellular cytokine staining (ICS) is tested for KRAS G12D, KRASG12V, KRAS G13D, G12C, KRAS WT, and no peptide.

The ability of the various mRNAs encoding KRAS mutations in combinationwith mRNA encoding constitutively active STING to generate T cellresponses is tested to allow for comparison of the effect of the STINGimmune potentiator on the various different KRAS constructs. Forexample, CD8 vaccine responses can be assessed by intracellular staining(ICS) for IFN-γ and/or TNF-α as described herein.

Example 8: Immune Potentiator mRNAs Enhance HPV Vaccine Responses

In this example, the potency and durability of responses to a humanpapillomavirus (HPV) E6/E7 mRNA-based vaccine used in combination withSTING, IRF3 or IRF7 immune potentiators were examined. A specific immuneresponse to human papillomavirus (HPV) in the cervical microenvironmentis known to play a key role in eradicating infection and eliminatingmutated cells. However, high-risk HPVs are known to modulate immunecells to create an immunosuppressive microenvironment (see e.g., Prata,T. T. et al. (2015) Immunology 146:113-121). Thus, an HPV vaccinationapproach that leads to a robust and durable immune response is highlydesirable.

The HPV vaccines used in this example were mRNA constructs encodingeither intracellular or soluble forms of HPV 16 antigens E6 and E7,referred to herein as iE6/E7 and sE6/E7, respectively. To create thesoluble format, a signal peptide required for secretion was fused to theN-terminal of the antigen. The sequence of the signal peptide wasderived from the Ig kappa chain V-III region HAH. Mice were immunizedintramuscularly with either the iE6/E7 or sE6/E7 mRNA vaccine (at a doseof 0.25 mg/kg) on days 0 and 14, combination with either a control mRNAcontruct (NTFIX), or a STING, IRF3 or IRF7 immune potentiator mRNAconstruct (at a dose of 0.25 mg/kg). The constitutively active STINGimmune potentiator contained a V155M mutation (mouse versioncorresponding to SEQ ID NO: 1). The constitutively active IRF3 immunepotentiator contained a S396D mutation (corresponding to SEQ ID NO: 12).The constitutively active IRF7 immune potentiator contained an internaldeletion and six point mutations (mouse version corresponding to SEQ IDNO: 18). The HPV vaccine construct and the immune potentiator constructwere coformulated in MC3 lipid nanoparticles.

At day 21 and 53, spleen cells and peripheral blood mononuclear cells(PBMC) from mice in each test group were restimulated ex vivo for 4hours at 37 degrees C. in the presence of GolgiPlug™ (containingBrefeldin A; BD Biosciences) with either: an E6 peptide pool (containing37 E6 peptides, the sequences of which are shown in SEQ ID NOs: 36-72),an E7 peptide pool (containing 22 E7 peptides, the sequences of whichare shown in SEQ ID NOs: 73-94), E6 single peptides (8 individualpeptides), E7 single peptides (7 individual peptides) or no peptides(control). CD8 vaccine responses were assessed by intracellular staining(ICS) for IFN-γ or TNF-α.

Representative ICS results for E7-specific responses by day 21 spleencells for IFN-γ and TNF-α are shown in FIG. 11A (IFN-γ) and FIG. 11B(TNF-α). Representative ICS results for E6-specific responses by day 21spleen cells for IFN-γ and TNF-α are shown in FIG. 12A (IFN-γ) and FIG.12B (TNF-α). The results in FIGS. 11A-11B and 12A-12B demonstrate thatCD8 vaccine responses (to both the intracellular and soluble antigenformat) were greatly enhanced when the STING, IRF3 or IRF7 immunepotentiators were co-formulated with the vaccine, with the E7 epitopebeing stronger and less variable than the E6 epitope and with thesoluble form of antigen being stronger than the intracellular form ofantigen. This enhanced CD8 vaccine responses by the immune potentiatorswas shown to be durable, as evidenced by the representative day 21versus day 53 E7-specific spleen cell IFN-γ ICS data shown in FIGS. 13Aand 13B, respectively. Similar results to the spleen cell data wereobserved for the PBMC experiments (data not shown).

The percentage of CD8b⁺ cells among the live CD45⁺ cells was alsoexamined. The results for day 21 versus day 53 spleen cells are shown inFIGS. 14A and 14B, respectively. The results demonstrate that the immunepotentiators (in particular the STING construct) expand the total CD8b⁺population on day 21 but not day 53.

The ability of the immune potentiator constructs to enhance the CD8vaccine response was further confirmed by E7-MHC1-tetramer staining.Representative results for day 21 versus day 53 spleen cells are shownin FIGS. 15A and 15B, respectively. The E7-MHC-1-tetramer stainingresults were consistent with the ICS results discussed above, althoughthey were more variable. As demonstrated in FIGS. 16A-16D, the majorityof the tetramer positive CD8 cells were found to have an “effectormemory” CD62L^(lo) phenotype. Comparison of day 21 versus day 53E7-tetramer⁺ CD8 cells demonstrated that this“effector-memory”CD62L^(lo) phenotype was maintained throughout thestudy. Additional staining experiments demonstrated that the immunepotentiators slightly reduced the % of total Foxp3⁺ Treg CD4 Tcells(data not shown) and did not change the % of CD138⁺ plasmablasts (datanot shown).

Example 9: Prophylactic or Therapeutic Vaccination with HPV Vaccine inCombination with STING Immune Potentiator Inhibits Tumor Growth

In this example, mice were treated with an HPV vaccine in combinationwith a STING immune potentiator either prior to, at the same time as, orafter challenge with TC1 tumor cells. TC-1 is an HPV16 E7-expressingmurine tumor model known in the art (see e.g., Bartkowiak et al. (2015)Proc. Natl. Acad. Sci. USA 112:E5290-5299). The HPV vaccines used inthis example were mRNA constructs encoding either intracellular orsoluble forms of HPV 16 antigens E6 and E7, referred to herein as iE6/E7and sE6/E7, respectively, as described in Example 8. The constitutivelyactive STING immune potentiator used in this example contained a V155Mmutation, as described in Example 8. The HPV vaccine construct and theimmune potentiator construct were coformulated in MC3 lipidnanoparticles. Certain mice were also treated with an immune checkpointinhibitor (either anti-CTLA-4 or anti-PD-1).

In a first set of experiments examining the prophylactic activity of theHPV+STING vaccination, C57/B6 mice were treated by intramuscularinjection with 0.5 mg/kg of the HPV+STING vaccine (encoding eithersE6/E7 or iE6/E7) on either (i) days −7 and −14, or (ii) days 1 and 8,followed by subcutaneous injection of 2×10⁵ TC1 cells on day 1. Certainmice were also treated on days 6, 9 and 12 with either anti-CTLA-4 oranti-PD-1. Representative results, reported as tumor volume over time,are shown in the graphs of FIGS. 17A-17C, wherein FIGS. 17A and 17B showdata for mice treated on days −14 and −7 with either sE6/E7 (FIG. 17A)or iE6/E7 (FIG. 17B) and FIG. 17C shows data for mice treated on days 1and 8 with sE6/E7. The results demonstrate that all of the mice treatedwith the HPV+STING vaccine (alone or in combination with immunecheckpoint inhibitors) showed complete inhibition of tumor growth overseveral weeks, as compared to the control mice (treated with the controlmRNA construct NTFIX, alone or in combination with an immune checkpointinhibitor). Thus, these experiments demonstrate that prophylacticvaccination (i.e., prior to or at the same time as tumor challenge) withthe HPV vaccine together with the STING immune potentiator is effectivein preventing growth of HPV-expressing tumor cells in vivo.

In a second set of experiments examining the therapeutic activity of theHPV+STING vaccination, C57/B6 mice were administered 2×10⁵ TC1 cellssubcutaneously on day 1, followed by treatment by intramuscularinjection with 0.5 mg/kg of the HPV+STING vaccine (encoding sE6/E7) ondays 8 and 15. Certain mice were also treated on days 13, 16 and 19 witheither anti-CTLA-4 or anti-PD-1. Representative results, reported astumor volume over time, are shown in the graphs of FIGS. 18A-18I. Theresults demonstrate that the mice treated with the HPV+STING vaccine(alone or in combination with immune checkpoint inhibitors) showed tumorregression (FIGS. 18A-18C), as compared to the control mice treated withthe control mRNA construct NTFIX, alone or in combination with an immunecheckpoint inhibitor (FIGS. 18D-18F) or the control mice treated withthe sE6/E7 construct in combination with the control DMXAA construct,alone or in combination with an immune checkpoint inhibitor (FIGS.18G-18I). Thus, these experiments demonstrate that therapeuticvaccination (i.e., subsequent to tumor challenge) with the HPV vaccinetogether with the STING immune potentiator is effective in inducingregression of HPV-expressing tumors in vivo.

Example 10. Determining Optimal Antigen:Immune Potentiator Mass Ratio inmRNA Vaccine Design

In this example, studies were performed in animals treated with anantigen of interest in combination with an immune potentiator atdifferent Ag:Immune Potentiator ratios, followed by examination of Tcell responses to the antigen, to determine optimal Ag:ImmunePotentiator ratios in enhancing the immune response to the antigen ofinterest.

In a first set of experiments, mice were treated with an MC38 vaccineencoding an ADR concatemer of three 25mer mutant peptides containingtumor neoepitopes derived from Adpgk, Dpagt1, and Reps1 (this vaccine isalso referred to herein as ADRvax), as described in Example 5, incombination with a constitutively active STING immune potentiatorconstruct. The constitutively active STING immune potentiator used inthis example contained a V155M mutation, as described in Example 1. TheADRvax and STING constructs were coformulated in a lipid nanoparticle(comprising Compound 25 (Cmp25)) at varying Ag:STING ratios, accordingto the study design summarized below in Table 13.

TABLE 13 STING Total Ag:STING Ag dose dose NTFIX mRNA Dosing Group ratio(μg) (μg) (μg) (μg) Vehicle Route Regimen 1 No Ag control 0 3 3 6 Cmp25IM Day 1, 15 2 1:1 3 3 0 3 5:1 0.6 2.4 4 10:1  0.3 2.7 5 20:1  0.15 2.856 1:0 (No STING) 0 3 7 1:1 5 5 0 10 8 1:0 (No STING) 0 5

Mice were dosed intramuscularly on days 1 and 15. At day 21, CD8⁺ spleencells from mice in each test group were restimulated ex vivo for 4 hoursat 37 degrees C. in the presence of GolgiPlug™ (containing Brefeldin A;BD Biosciences) with either wild-type or mutant MC38 ADR peptides (1μg/ml per peptide, pooled) and CD8 vaccine responses were assessed byintracellular staining (ICS) for IFN-γ or TNF-α. Representative ICSresults for MC38 ADR-specific responses by day 21 CD8⁺spleen cells forIFN-γ are shown in FIG. 19 and for TNF-α are shown in FIG. 20.Additionally, CD8 vaccine responses to each of the three individualepitopes within ADRvax (i.e., peptides Adpk1, Reps1 and Dpagt1) werealso assessed by ICS for IFN-γ following stimulation with the individualepitopes. The results are shown in FIG. 21A (for peptide Adpk1), FIG.21B (for peptide Reps1) and FIG. 21C (for peptide Dpagt1).

The results demonstrate that all Ag:STING ratios tested (ranging from1:1 to 20:1) showed an adjuvant effect of STING as compared to control.For the ADRvax antigen as a whole, the optimal Ag:STING ratio was foundto be 5:1. For the individual peptide epitopes within ADRvax, theoptimal Ag:STING ratio for the Adpgk1 peptide was 5:1, whereas theoptimal Ag:STING ratio for the Reps1 peptide was 10:1 (the responses tothe third peptide, Dpagt1, were very low with or without STING,consistent with it being a non-dominant epitope as was known in theart). STING was also found to increase the total percentage of CD8+cells among CD45+ T cells, with dose responses observed (data not shown)and was found to increase the total percentage of CD62L cells amongCD44hi CD8+ cells (effector/memory subset), with dose responses observed(data not shown). Furthermore, results obtained from PBMC cells wereconsistent with the spleen cell results (data not shown). Thus, theseexperiments confirmed the ability of STING to act as an immunepotentiator in enhancing immune responses against the ADRvax antigenand, moreover, demonstrated the determination of an optimal Ag:ImmunePotentiator ratio for treatment, with ratios other than 1:1 being foundto be most optimal (e.g., ratios of 5:1 or 10:1 being more effectivethan 1:1). The results further indicate that the optimal Ag:ImmunePotentiator ratio may differ depending on the particular antigen ofinterest used.

In a second set of experiments, non-human primates were treated with anHPV vaccine encoding intracellular E6/E7 (iE6/E7), as described inExample 8, in combination with the constitutively active STING immunepotentiator construct at varying Ag:STING ratios (lipid nanoparticlescomprising Compound 25), according to the study design summarized belowin Table 14:

TABLE 14 Ag:STING μg μg μg Total Ag Group Treatment Ratio Ag STING NTFIXn Dose 1 STING only — — 100 — 3 100 μg 2 Ag:STING 1:1 50 50 — 3 Ag:STING5:1 83.33 16.67 — 4 Ag:STING 10:1  90.9 9.09 5 Ag only — 90 — 10No clinical findings were observed 24 hours after the first dose(administered intramuscularly), indicating no injection site reactionsand that the initial treatment was received safely. After an initialdosing on Day 1, animals have a two week recover period and then aregiven a second dose at day 14, followed by another two week recoveryperiod. Further safety analysis is determined by clinical pathology(clinical chemistry, hematology and coagulation) at days 2, 16 and 30.Anti-antibody and ELISpot analysis or ICS for IFN-γ for CD4 and CD8cells are performed to assess enhancement of immune responses to the HPVvaccine by STING at the varying ratios tested.

In a third set of experiments, a model concatemeric antigen using knownmurine epitopes was tested in mice in combination with theconstitutively active STING immune potentiator at varying ratios. Theconcatemeric antigen, referred to herein as CA-132, comprises 20 knownmurine epitopes thought to be presented on MHC Class I and Class IIantigens of the CB6 mouse. These epitopes were sourced from the IEDB.orgwebsite, a public database of epitopes sourced from the literature.Class I epitopes are expected to be presented on MHC Class I moleculesand trigger a CD8+ response, while Class II epitopes are expected to bepresented on MHC Class II molecules and trigger CD4+ T cell responses.The CA-132 antigen construct encodes both Class I and Class II epitopes,allowing for assessment of both CD4 and CD8 T cell responses. Moreover,it is believed that inclusion of Class II epitopes in the concatemericantigen (thus triggering a CD4 response) helps induce a stronger CD8 Tcell response. Thus, the approach to the design of the CA-132 antigencan also be used in the design of other concatemeric antigen constructs.

The CA-132 antigen construct and STING immune potentiator construct werecoformulated in lipid nanoparticles comprising Compound 25 andadministered intramuscularly to CB6 mice at the following dosages:CA-132 alone at 1 μg, 3 μg or 10 μg, STING alone at 3 μg, CA-132+STINGat either 3 μg each or 1 μg each (1:1 ratio), CA-132 at 3 μg and STINGat 1 μg (Ag:STING ratio of 3:1) or CA-132 at 1 μg and STING at 3 μg(Ag:STING ratio of 1:3). Antigen-specific T cell responses to the ClassI epitopes within the CA-132 antigen construct were examined by ELISpotanalysis for IFN-γ, the results of which are shown in FIG. 22. Theresults demonstrated an increase in IFN-γ responses to the Class Iepitopes when formulated with STING.

In a fourth series of experiments, the HPV vaccine model described inExample 8 was used to study the effect of varying ratios of E6/E7antigen to constitutively active STING immune potentiator. Mice wereimmunized intramuscularly with the iE6/E7 mRNA vaccine (3 μg or 5 μg) incombination with the V155M constitutively active STING immunepotentiator mRNA construct at Ag:STING ratios of 1:1, 5:1, 10:1, 20:1 or0.4:1. The HPV vaccine construct and the immune potentiator constructwere coformulated in MC3 lipid nanoparticles. HPV vaccine or STING incombination with only a control mRNA (NTFIX) were used as controls.

At day 21, spleen cells from mice in each test group were restimulatedex vivo for 4 hours at 37 degrees C. in the presence of GolgiPlug™(containing Brefeldin A; BD Biosciences) with an E7 peptide pool(described in Example 8). CD8 vaccine responses were assessed byintracellular staining (ICS) for IFN-γ. The results are shown in FIG.23A. The results demonstrate that STING enhanced the antigen-specific Tcell responses at all Ag:STING ratios tested. The largest enhancementwas observed for the mice treated with the higher dose of antigen (5 μg)at a 1:1 ratio with STING and for the mice treated at an Ag:STING ratioof 0.4:1 (3 μg Ag to 7 μg STING).

The ability of STING to enhance the CD8 vaccine response in the HPVmodel at various Ag:STING ratios tested was further confirmed byH2-Kb/E7 peptide-tetramer staining. Representative results for day 21spleen cells are shown in FIG. 23B. The E7-MHC-1-tetramer stainingresults were consistent with the ICS results discussed above, althoughthey were more variable.

In summary, these studies confirmed the ability of the STING immunepotentiator construct to enhance immune responses to an antigen ofinterest and demonstrated the determination of optimal Ag:STING ratiosfor treatment.

Example 11: Immune Potentiation by STING in Non-Human Primates

In this example, non-human primates (cynomolgus monkeys) were treatedwith mRNAs encoding an HPV vaccine in combination with a STING immunepotentiator, followed by assessment of antigen-specific T cell andantibody responses. The HPV vaccine construct used in this example isdescribed in Example 8. The constitutively active STING immunepotentiator construct used in this example contained a V155M mutation,as described in Example 8. The HPV vaccine construct and the immunepotentiator mRNA constructs were coformulated in lipid nanoparticlescomprising: Compound 25:Cholesterol:DSPC:PEG-DMG, at ratios of50:38.5:10:1.5, respectively. Different ratios of STING:Ag were tested.Control animals were treated with mRNAs encoding either the HPV antigensalone or the STING immune potentiator alone.

Fifteen male cynomolgus monkeys, 2-5 years old and weighing 2-5 kg, weretreated according to the study design shown below in Table 15:

TABLE 15 Total mRNA STING HPV Ag Group Desc. Ratio (μg) NTFIX (μg) (μg)n 1 Ag only 100 10 90 3 2 STING only 100 100 0 3 3 STING:Ag 1:1 100 5050 3 4 STING:Ag 1:5 100 17 83 3 5 STING:Ag  1:10 100 9 91 3

A pre-dose sample of PBMCs were collected on day −4, followed bytreatment of the animals intramuscularly with the mRNA LNPs on day 1 andday 15. A post-dose sample of PBMCs was collected on day 29. No toxicityor other major clinical observations were noted during the study,indicating the mRNA LNPs were well-tolerated.

To examine the ability of the STING immune potentiator to enhanceantigen-specific CD8+ T cell responses, intracellular cytokine staining(ICS) for TNFα and IL-2 was conducted. PBMCs were stimulated ex vivowith the HPV16 E6 peptide pool or the HPV16 E7 peptide pool for 6 hoursat 37° C. Stimulation with PMA/ionomycin was used as a positive controland stimulation with medium alone was used as a negative control.

Representative results for ICS for TNFα are shown in FIGS. 24A-24C,wherein FIG. 24A shows results for ex vivo stimulation with the E6peptide pool, FIG. 24B shows the results for ex vivo stimulation withthe E7 peptide pool and FIG. 24C shows the results for ex vivostimulation with the medium control. No increase in TNFα+ CD8 T cellfrequency was observed between the pre- and post-dose group immunizedwith antigen alone (Group 1). Immunization with STING treatment alone(Group 2) had a marginal effect on TNFα+ CD8 T cell frequency. Incontrast, groups immunized with STING+Ag (Groups 3, 4, 5) showed asignificant increase in antigen-specific TNFα+ CD8 T cells. Furthermore,Group 5, which was immunized with a “matching” antigen dose of STING:Ag(1:10 ratio), showed a significant increase in antigen-specific TNFα+CD8 T cells when compared to the Group 1 and Group 2 controls.

Representative results for ICS for IL-2 are shown in FIGS. 25A-25C,wherein FIG. 25A shows results for ex vivo stimulation with the E6peptide pool, FIG. 25B shows the results for ex vivo stimulation withthe E7 peptide pool and FIG. 25C shows the results for ex vivostimulation with the medium control. A moderate increase in IL-2+ CD8 Tcell frequency between the pre- and post-dose was observed in allimmunized animals (Groups 1-5). However, the increase in IL-2+ CD8 Tcells was most detectable in the groups treated with STING:Ag at ratiosof 1:1 and 1:5 (Groups 3 and 4), whereas animals treated with STING:Agat a 1:10 ratio did not exhibit increased IL-2+ CD8 T cells as comparedto controls. The increase in IL-2 is consistent with the known abilityof subsets of T cells to secrete IL-2 during active T cell responses.

To examine the effect of STING:Ag treatment in the NHPs onantigen-specific antibody responses, E6-specific and E7-specific ELISAswere performed. Plates were coated with either recombinant E6 (Prospec;#HPV-005 His HPV16 E6) or recombinant E7 (ProteinX; #2003207 His HPV16E7). A mouse anti-E6 monoclonal antibody from Alpha DiagnosticsInternational (#HPV16E6 1-M) was used as a positive control. A mouseanti-E7 monoclonal antibody from Fisher/Life Technologies (#280006-EA)was used as a positive control. An anti-mouse IgG-HRP antibody fromJackson ImmunoResearch (#715-035-150) was used as the secondary antibodyfor the positive controls. Anti-monkey IgG-HRP from Abcam (#ab112767)was used as the secondary antibody for the NHP serum.

Plates were coated with recombinant E6 or E7 (500 ng/well; 100 μl/well)at 4° C. overnight and then blocked with TBS SuperBlock for 1 hour atroom temperature. Primary antibody was added (100 μl/well) and incubatedfor 1 hour at room temperature. Positive control antibodies wereserially diluted. NHP serum was diluted 1:5000. After washing, secondaryantibody was added (100 μl/well) and incubated for 1 hour at roomtemperature. Positive control anti-mouse IgG-HRP was diluted 1:5000. Forthe NHP serums, anti-monkey IgG-HRP was diluted 1:30,000. Color wasdeveloped for 5 minutes (anti-E6) or for 10 minutes (anti-E7), thenstopped and read at 450 nm.

Representative results for anti-HPV16 E6 IgG are shown in FIG. 26.Representative results for anti-HPV16 E7 IgG are shown in FIG. 27. Theresults for both anti-E6 and anti-E7 demonstrate that treatment of theanimals with STING:Ag, particularly at ratios of 1:5 and 1:10 led toincreased antigen-specific antibody responses.

To further study the antigen-specific IgG response, further ELISAstudies were performed using a two-fold dilution series for day 25serums. As shown in FIG. 28, the two-fold dilution series for theanimals treated at a 1:10 STING:Ag ratio exhibited a clear enhancementin the levels of anti-HPV16 E6-specific IgG antibodies, as compared toanimals treated with the HPV vaccine alone. Calculated titer values fromthese ELISA studies with the day 25 serum two-fold dilution series foranti-E6 IgG and anti-E7 IgG are shown in FIGS. 29A and 29B,respectively. The calculated titer values, particularly for the anti-E6specific response, confirm the enhancement by the STING immunepotentiator, with the 1:10 STING:Ag ratio showing the greatestenhancement.

Accordingly, the results described herein for the non-human primatestudy confirm that STING immunopotentiates antigen-specific T cell andantibody responses against an mRNA vaccine antigen in vivo.

Example 12: Immunogenicity of Various KRAS-STING Vaccine Formats inHLA*A11 Transgenic Mice

In this example, to examine the effect of the STING immune potentiatormRNA construct on immune responses to various different forms of themutant KRAS peptide antigen mRNA constructs, HLA*A*11:01 Tg mice(Taconic, strain 9660F, n=3) were administered mRNA encoding variousdifferent forms of mutated KRAS peptide antigen mRNA constructs incombination with a STING immune potentiator mRNA construct as follows:mRNA encoding mutated KRAS in combination with STING administered ondays 0 and 14, animals sacrificed on day 21. Mice were aged 6-9 weeks atday 0. mRNA was administered to the animals at a dose of 0.5 mg/kg (10ug per 20-g animal). The KRAS and STING constructs are administered at a5:1 ratio (Ag:STING).

The types of mutated KRAS constructs tested were as follows: (i) mRNAencoding a single mutant KRAS 25mer peptide antigen containing eitherthe G12D, G12V, G13D or G12C mutation (“monomer”); (ii) mRNA encoding aconcatemer of three 25mer peptide antigens (thus creating a 75mer), oneof each containing the G12D, G12V and G13D mutations (“KRAS-3MUTconcatemer”); (iii) mRNA encoding a concatemer of four 25mer peptideantigens (thus creating a 100mer), one of each containing the G12D,G12V, G13D and G12C mutations (“KRAS-4MUT concatemer”); or (iv) fourseparate mRNAs coadministered together, each encoding a single mutantKRAS 25mer peptide antigen containing either the G12D, G12V, G13D orG12C mutation (“pooled monomers”). The amino acid and nucleotidesequences of the constructs are as described in Example 7. An A11-viralepitope concatemer antigen was also tested in combination with STING ora control mRNA (NTFIX) (“validated A11 Ag”).

The test groups are shown in Table 16 as follows:

TABLE 16 Test/Control Immune Dosing TEST group Group MaterialPotentiator Vehicle Route Regimen KRAS-MUT 1 KRAS G12D STING (V155M)Compound 25 IM Day 1, 14 Monomer 2 KRAS G12V STING (V155M) Compound 25IM Day 1, 14 3 KRAS G13D STING (V155M) Compound 25 IM Day 1, 14 4 KRASG12C STING (V155M) Compound 25 IM Day 1, 14 KRAS-MUT 5 KRAS-3MUT STING(V155M) Compound 25 IM Day 1, 14 Concatemer 6 KRAS-4MUT STING (V155M)Compound 25 IM Day 1, 14 7 KRAS-4MUT.var1 STING (V155M) Compound 25 IMDay 1, 14 Pooled 8 G12D + G12V + STING (V155M) Compound 25 IM Day 1, 14Monomers G12C + G13D Validated 9 A11-Viral epitope STING (V155M)Compound 25 IM Day 1, 14 A11 Ags concatemer 10 A11-Viral epitope NTFIXCompound 25 IM Day 1, 14 concatemer

In a first set of experiments to evaluate antigen-specific CD8+ T cellresponses to the KRAS antigens, day 21 spleen cells from the mice wererestimulated ex vivo with KRAS monomer peptides (2 ug/ml per peptide)for 5 hours at 37 degrees Celsius in the presence of GolgiPlug(Brefeldin A). Intracellular cytokine staining (ICS)(IFN-γ) wasperformed for KRAS G12D (aa*7/8-16), KRAS G12V (aa*7/8-16), KRAS G13D(aa*7/8-16), G12C (aa*7/8-16), KRAS WT (aa*7/8-16) and no peptide.

The ICS results for KRAS-G12V-specific responses are shown in FIG. 30.The ICS results for KRAS-G12D-specific responses are shown in FIG. 31.These results demonstrate that anti-KRAS-G12V and anti-KRAS-G12Dspecific CD8+ T cells were detected in mice immunized with thecorresponding KRAS-STING vaccine (monomer or concatemer) andrestimulated with the cognate peptide. Comparable % IFN-gamma positiveCD8+ T cells were seen when the KRAS mutations were administered to themice as a monomer or as concatemers. The responses observed with G12Vwere stronger than the responses observed with G12D. In this experiment,anti-KRAS G12C and anti-KRAS G13D responses were not observed (data notshown).

In a second set of experiments to evaluate antigen-specific CD8+ T cellresponses to KRAS antigens, day 21 spleen cells from the mice wereco-cultured with HLA*A11-expressing target cells (Cos7-A11 cells) thathad been pulsed with the corresponding KRAS peptides (G12V, G12D or WTcontrol), followed by ICS (IFN-γ). The Cos7-A11 co-culture results forKRAS-G12V-specific responses are shown in FIG. 32. The Cos7-A11co-culture results for KRAS-G12D-specific responses are shown in FIG.33. These results demonstrate that anti-KRAS-G12V and anti-KRAS-G12Dspecific CD8+ T cell responses were detected in mice immunized with thecorresponding KRAS-STING vaccine (monomer or concatemer) andrestimulated with the A11+ expressing cell line pulsed with G12V orG12D. Thus, the results in this second set of experiments with respectto detection of antigen-specific CD8+ T cell responses to the KRASantigens were very similar to the results from the first set ofexperiments using restimulation with cognate peptides.

Finally, the ability of STING to potentiate antigen-specific response toknown A*11-restricted viral epitopes was evaluated using day 21 spleencells from the mice immunized with an A11-viral epitope concatemer.Eight viral epitopes (EBV BRLF1, FLU, HIV NEF, EBV, HBV core antigen,HCV, CMV and BCL-2L1) (25 amino acids each) were concatemerized andencoded by mRNA for use as an antigen in combination with STING in theA11-transgenic mice (treatment group 9 in Table 16). The A11-viralepitope concatemer was also co-administered with an NTFIX control mRNA(treatment group 10 in Table 16). Five of the eight epitopes (EBV BRLF1,FLU, HIV NEF, EBV, HBV core antigen) were validated A11 binders withrelatively low predicted IC50s; the other three epitopes (HCV, CMV andBCL-2L1) had more moderate predicted affinities for A11 but have notbeen experimentally validated. The amino acid sequences for the viralepitopes, as well as their IC50s, are shown below in Table 17:

TABLE 17 Literature Gene Peptide ann_IC50 % rank validation EBVATIGTAMYK (SEQ ID NO: 226) 6.03 0.2 Y BRLF1 FLU SIIPSGPLK (SEQ ID NO:227) 5 0.25 Y HIV NEF AVDLSHFLK (SEQ ID NO: 228) 20.31 0.25 Y EBVAVFDRKSDAK (SEQ ID NO: 229) 55.63 0.5 Y HBV YVNVNMGLK (SEQ ID NO: 230)69.82 0.5 Y core antigen HCV RVCEKMALY (SEQ ID NO: 231) 304.91 1.3 CMVKLGGALQAK (SEQ ID NO: 232) 736.59 1.6

Day 21 spleen cells were restimulated ex vivo with the individual A*11viral epitopes, followed by ICS (IFN-γ and TNF-α), to detectantigen-specific CD8+ T cell responses. Antigen-specific CD8+ T cellresponses were observed for four out of the eight viral epitopes (EBV,EBV BRLF1, FLU and HIV NEF) and, as shown in FIG. 34, STING potentiatedT cell responses for these four viral epitopes.

A repeat study was performed in HLA*A11 transgenic mice using theKRAS-4MUT concatemer, at either a low dose (10 μg) or a high dose (30μg), in combination with the STING immune potentiator mRNA at anAg:STING ratio of 5:1. Significant enhancement of G12V-specific CD8 Tcell responses by the STING immune potentiator construct was againobserved, with the greatest enhancement being seen at the higher dose ofantigen tested (30 μg).

Accordingly, the results described herein for HLA*A11 transgenic micedemonstrate that STING immunopotentiates antigen-specific T cellanti-KRAS responses, as well as anti-viral responses to otherA11-restricted viral antigens, and is able to immunopotentiate responsesto vaccine antigens in various formats (monomers and concatemers).

Other Embodiments

It is to be understood that while the present disclosure has beendescribed in conjunction with the detailed description thereof, theforegoing description is intended to illustrate and not limit the scopeof the present disclosure, which is defined by the scope of the appendedclaims. Other aspects, advantages, and alterations are within the scopeof the following claims.

All references described herein are incorporated by reference in theirentireties.

SEQUENCE LISTING SUMMARY SEQ ID NO: SEQUENCE 1MPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEELRHIHSRYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALNILLGLKGLAPAEISAVCEKGNFNMAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDHAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDRLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLRTDFS(huSTING(V155M); no epitope tag) 2MPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEELRHIHSRYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALNILLGLKGLAPAEISAVCEKGNFNVAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDHAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDtLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLRTDFS (HuSTING(R284T); no epitope tag) 3MPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEELRHIHSRYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALNILLGLKGLAPAEISAVCEKGNFNVAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDHAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDmLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLRTDFS (hu STING(R284M); no epitope tag) 4MPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEELRHIHSRYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALNILLGLKGLAPAEISAVCEKGNFNVAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDHAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDkLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLRTDFS (Hu STING(R284K); no epitope tag) 5MPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEELRHIHSRYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALNILLGLKGLAPAEISAVCEKGNFsVAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDHAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDRLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLRTDFS (HuSTING(N154S); no epitope tag) 6MPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEELRHIHSRYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALNILLGLKGLAPAEISAICEKGNFNVAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDHAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDRLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLRTDFS (HuSTING(V147L); no epitope tag) 7MPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEELRHIHSRYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALNILLGLKGLAPAEISAVCEKGNFNVAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDHAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDRLEQAKLFCRTLEDILADAPESQNNCRLIAYQqPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLRTDFS (Hu STING(E315Q); no epitope tag) 8MPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEELRHIHSRYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALNILLGLKGLAPAEISAVCEKGNFNVAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDHAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDRLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLaTDFS (Hu STING(R375A); no epitope tag) 9MPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEELRHIHSRYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALNILLGLKGLAPAEISALCEKGNFSMAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDHAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDRLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLRTDFS (Hu STING(V147L/N154S/V155M); no epitope tag) 10MPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEELRHIHSRYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALNILLGLKGLAPAEISALCEKGNFSMAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDHAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDMLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLRTDFS (Hu STING(R284M/V147L/N154S/V155M); no epitope tag)11METPKPRILPWLVSQLDLGQLEGVAWLDESRTRFRIPWKHGLRQDAQMADFGIFQAWAEASGAYTPGKDKPDVSTWKRNFRSALNRKEVLRLAADNSKDPYDPHKVYEFVTPGARDFVHLGASPDTNGKSSLPHSQENLPKLFDGLILGPLKDEGSSDLAIVSDPSQQLPSPNVNNFLNPAPQENPLKQLLAEEQWEFEVTAFYRGRQVFQQTLFCPGGLRLVGSTADMTLPWQPVTLPDPEGFLTDKLVKEYVGQVLKGLGNGLALWQAGQCLWAQRLGHSHAFWALGEELLPDSGRGPDGEVHKDKDGAVFDLRPFVADLIAFMEGSGHSPRYTLWFCMGEMWPQDQPWVKRLVMVKVVPTCLKELLEMAREGGASSLKTVDLHIDNSQPISLTSDQYKAYLQDLVEDMDFQATGNI (super mouse IRF3 S396D; noepitope tag) 12MGTPKPRILPWLVSQLDLGQLEGVAWVNKSRTRFRIPWKHGLRQDAQQEDFGIFQAWAEATGAYVPGRDKPDLPTWKRNFRSALNRKEGLRLAEDRSKDPHDPHKIYEFVNSGVGDFSQPDTSPDTNGGGSTSDTQEDILDELLGNMVLAPLPDPGPPSLAVAPEPCPQPLRSPSLDNPTPFPNLGPSENPLKRLLVPGEEWEFEVTAFYRGRQVFQQTISCPEGLRLVGSEVGDRTLPGWPVTLPDPGMSLTDRGVMSYVRHVLSCLGGGLALWRAGQWLWAQRLGHCHTYWAVSEELLPNSGHGPDGEVPKDKEGGVFDLGPFIVDLITFTEGSGRSPRYALWFCVGESWPQDQPWTKRLVMVKVVPTCLRALVEMARVGGASSLENTVDLHIDNSHPLSLTSDQYKAYLQDLVEGMDFQGPGET (super human IRF3S396D; no epitope tag) 13MALAPERAAPRVLFGEWLLGEISSGCYEGLQWLDEARTCFRVPWKHFARKDLSEADARIFKAWAVARGRWPPSSRGGGPPPEAETAERAGWKTNFRCALRSTRRFVMLRDNSGDPADPHKVYALSRELCWREGPGTDQTEAEAPAAVPPPQGGPPGPFLAHTHAGLQAPGPLPAPAGDKGDLLLQAVQQSCLADHLLTASWGADPVPTKAPGEGQEGLPLTGACAGGPGLPAGELYGWAVETTPSPgpqpaalttgeaaapesphqaepylspspsactavqepspgaldvtimykgrtvlqkvvghpsctflygppdpavratdpqqvafpspaelpdqkqlryteellrhvapglhlelrgpqlwarrmgkckvywevggppgsaspstpacllprncdtpifdfrvffqelvefrarqrrgsprytiylgfgqdlsagrpkekslvlvklepwlcrvhlegtqrEGVSSLDSSSLSLCLSSANSLYDDIECFLMELEQPA(Wild-type Hu IRF7 isoform A; P037 without epitope tag) 14MALAPERAAPRVLFGEWLLGEISSGCYEGLQWLDEARTCFRVPWKHFARKDLSEADARIFKAWAVARGRWPPSSRGGGPPPEAETAERAGWKTNFRCALRSTRRFVMLRDNSGDPADPHKVYALSRELCWREGPGTDQTEAEAPAAVPPPQGGPPGPFLAHTHAGLQAPGPLPAPAGDKGDLLLQAVQQSCLADHLLTASWGADPVPTKAPGEGQEGLPLTGACAGGPGLPAGELYGWAVETTPSPgpqpaalttgeaaapesphqaepylspspsactavqepspgaldvtimykgrtvlqkvvghpsctflygppdpavratdpqqvafpspaelpdqkqlryteellrhvapglhlelrgpqlwarrmgkckvywevggppgsaspstpacllprncdtpifdfrvffqelvefrarqrrgsprytiylgfgqdlsagrpkekslvlvklepwlcrvhlegtqrEGVSSLDSSdLdLCLSSANSLYDDIECFLMELEQPA(constitutively active Hu IRF7 S477D/S479D; P033 without epitope tag) 15MALAPERAAPRVLFGEWLLGEISSGCYEGLQWLDEARTCFRVPWKHFARKDLSEADARIFKAWAVARGRWPPSSRGGGPPPEAETAERAGWKTNFRCALRSTRRFVMLRDNSGDPADPHKVYALSRELCWREGPGTDQTEAEAPAAVPPPQGGPPGPFLAHTHAGLQAPGPLPAPAGDKGDLLLQAVQQSCLADHLLTASWGADPVPTKAPGEGQEGLPLTGACAGGPGLPAGELYGWAVETTPSPgpqpaalttgeaaapesphqaepylspspsactavqepspgaldvtimykgrtvlqkvvghpsctflygppdpavratdpqqvafpspaelpdqkqlryteellrhvapglhlelrgpqlwarrmgkckvywevggppgsaspstpacllprncdtpifdfrvffqelvefrarqrrgsprytiylgfgqdlsagrpkekslvlvklepwlcrvhlegtqrEGVSSLDdSdLSdCLSSANSLYDDIECFLMELEQPA(constitutively active Hu IRF7 S475D/S477D/L480D; P034 without epitopetag) 16MALAPERAAPRVLFGEWLLGEISSGCYEGLQWLDEARTCFRVPWKHFARKDLSEADARIFKAWAVARGRWPPSSRGGGPPPEAETAERAGWKTNFRCALRSTRRFVMLRDNSGDPADPHKVYALSRELCWREGPGTDQTEAEAPAAVPPPQGGPPGPFLAHTHAGLQAPGPLPAPAGDKGDLLLQAVQQSCLADHLLTASWGADPVPTKAPGEGQEGLPLTGACAGGPGLPAGELYGWAVETTPSPgpqpaalttgeaaapesphqaepylspspsactavqepspgaldvtimykgrtvlqkvvghpsctflygppdpavratdpqqvafpspaelpdqkqlryteellrhvapglhlelrgpqlwarrmgkckvywevggppgsaspstpacllprncdtpifdfrvffqelvefrarqrrgsprytiylgfgqdlsagrpkekslvlvklepwlcrvhlegtqrEGVSSLDdddLdLCLdSANdLYDDIECFLMELEQPA(constitutively active Hu IRF7 S475D/S476D/S477D/S479D/S483D/S487D; P035without epitope tag) 17MALAPERAAPRVLFGEWLLGEISSGCYEGLQWLDEARTCFRVPWKHFARKDLSEADARIFKAWAVARGRWPPSSRGGGPPPEAETAERAGWKTNFRCALRSTRRFVMLRDNSGDPADPHKVYALSRELCWREGPGTDQTEAEAPAAVPPPQGGPPGPFLAHTHAGLQAPGPLPAPAGDKGDLLLQAVQQSCLADHLLTASWGADPVPTKAPGEGQEGLPLTGACAGGPGLPAGELYGWAVETTPSPEGVSSLDSSSLSLCLSSANSLYDDIECFLMELEQPA (constitutivelyactive truncated Hu IRF7 1-246 + 468-503; P032 without epitope tag) 18MALAPERAAPRVLFGEWLLGEISSGCYEGLQWLDEARTCFRVPWKHFARKDLSEADARIFKAWAVARGRWPPSSRGGGPPPEAETAERAGWKTNFRCALRSTRRFVMLRDNSGDPADPHKVYALSRELCWREGPGTDQTEAEAPAAVPPPQGGPPGPFLAHTHAGLQAPGPLPAPAGDKGDLLLQAVQQSCLADHLLTASWGADPVPTKAPGEGQEGLPLTGACAGGPGLPAGELYGWAVETTPSPEGVSSLDdddLdLCLdSANdLYDDIECFLMELEQPA (constitutivelyactive truncated Hu IRF7 1-246 + 468-503 plusS475D/S476D/S477D/S479D/S483D/S487D; P036 without epitope tag) 19MALAPERAAPRVLFGEWLLGEISSGCYEGLQWLDEARTCFRVPWKHFARKDLSEADARIFKAWAVARGRWPPSSRGGGPPPEAETAERAGWKTNFRCALRSTRRFVMLRDNSGDPADPHKVYALSRELCWREGPGTDQTEAEAPAAVPPPQgpqpaalttgeaaapesphqaepylspspsactavqepspgaldvtimykgrtvlqkvvghpsctflygppdpavratdpqqvafpspaelpdqkqlryteellrhvapglhlelrgpqlwarrmgkckvywevggppgsaspstpacllprncdtpifdfrvffqelvefrarqrrgsprytiylgfgqdlsagrpkekslvlvklepwlcrvhlegtqrEGVSSLDSSSLSLCLSSANSLYDDIECFLMELEQPA(truncated Hu IRF7 1-151 + 247-503; P038 without epitope tag; nullmutation) 20MGGPPGPFLAHTHAGLQAPGPLPAPAGDKGDLLLQAVQQSCLADHLLTASWGADPVPTKAPGEGQEGLPLTGACAGGPGLPAGELYGWAVETTPSPgpqpaalttgeaaapesphqaepylspspsactavqepspgaldvtimykgrtvlqkvvghpsctflygppdpavratdpqqvafpspaelpdqkqlryteellrhvapglhlelrgpqlwarrmgkckvywevggppgsaspstpacllprncdtpifdfrvffqelvefrarqrrgsprytiylgfgqdlsagrpkekslvlvklepwlcrvhlegtqrEGVSSLDSSSLSLCLSSANSLYDDIECFLMELEQPA(truncated Hu IRF7 152-503; P039 without epitope tag; null mutation) 21TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACC (5′ UTR) 22TGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC (3′ UTR) 23TGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCCAAACACCATTGTCACACTCCATCCCCCCAGCCCCTCCTCCCCTTCCTCCATAAAGTAGGAAACACTACATGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC (3′ UTR with mi-122 and mi-142.3p sites) 24GSGATNFSLLKQAGDVEENPGP (2A peptide amino acid sequence) 25GGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCT(Nucleotide sequence encoding 2A peptide) 26TCCGGACTCAGATCCGGGGATCTCAAAATTGTCGCTCCTGTCAAACAAACTCTTAACTTTGATTTACTCAAACTGGCTGGGGATGTAGAAAGCAATCCAGGTCCACTC (Nucleotide sequence encoding 2Apeptide) 27GACAGUGCAGUCACCCAUAAAGUAGAAAGCACUACUAACAGCACUGGAGGGUGUAGUGUUUCCUACUUUAUGGAUGAGUGUACUGUG (miR-142) 28 UGUAGUGUUUCCUACUUUAUGGA (miR-142-3p) 29UCCAUAAAGUAGGAAACACUACA (miR-142-3p binding site) 30CAUAAAGUAGAAAGCACUACU (miR-142-5p) 31 AGUAGUGCUUUCUACUUUAUG (miR-142-5pbinding site) 32 AACGCCAUUAUCACACUAAAUA (miR-122-3p) 33UGGAGUGUGACAAUGGUGUUUG (miR-122-5p) 34 UAGCUUAUCAGACUGAUGUUGA(miR-21-5p) 35 CAACACCAGUCGAUGGGCUGU (miR-21-3p) 36 MKLVVVGADGVGKSAL(KRAS(G12D)15mer) 37 MKLVVVGAVGVGKSAL (KRAS(G12V)15mer) 38MLVVVGAGDVGKSALT (KRAS(G13D)15mer) 39 MTEYKLVVVGADGVGKSALTIQLIQ(KRAS(G12D)25mer) 40 MTEYKLVVVGAVGVGKSALTIQLIQ (KRAS(G12V)25mer) 41MTEYKLVVVGAGDVGKSALTIQLIQ (KRAS(G13D)25mer) 42MKLVVVGADGVGKSALKLVVVGADGVGKSALKLVVVGADGVGKSAL (KRAS(G12D)15mer^3) 43MKLVVVGAVGVGKSALKLVVVGAVGVGKSALKLVVVGAVGVGKSAL (KRAS(G12V)15mer^3) 44MLVVVGAGDVGKSALTLVVVGAGDVGKSALTLVVVGAGDVGKSALT (KRAS(G13D)15mer^3) 45MTEYKLVVVGADGVGKSALTIQLIQMTEYKLVVVGADGVGKSALTIQLIQMTEYKLVVVGADGVGKSALTIQLIQ(KRAS(G12D)25mer^3) 46MTEYKLVVVGAVGVGKSALTIQLIQMTEYKLVVVGAVGVGKSALTIQLIQMTEYKLVVVGAVGVGKSALTIQLIQ(KRAS(G12V)25mer^3) 47MTEYKLVVVGAGDVGKSALTIQLIQMTEYKLVVVGAGDVGKSALTIQLIQMTEYKLVVVGAGDVGKSALTIQLIQ(KRAS(G13D)25mer^3) 48MPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEELRHIHSRYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALNILLGLKGLAPAEISAVCEKGNFNMAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDHAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDRLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLRTDFSATNFSLLKQAGDVEENPGPMKLVVVGADGVGKSAL (KRAS(G12D)15mer_nt.STING(V155M)) 49MPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEELRHIHSRYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALNILLGLKGLAPAEISAVCEKGNFNMAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDHAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDRLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLRTDFSATNFSLLKQAGDVEENPGPMKLVVVGAVGVGKSAL (KRAS(G12V)15mer_nt.STING(V155M) 50MPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEELRHIHSRYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALNILLGLKGLAPAEISAVCEKGNFNMAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDHAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDRLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLRTDFSATNFSLLKQAGDVEENPGPMLVVVGAGDVGKSALT (KRAS(G13D)15mer_nt.STING(V155M) 51MPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEELRHIHSRYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALNILLGLKGLAPAEISAVCEKGNFNMAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDHAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDRLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLRTDFSATNFSLLKQAGDVEENPGPMTEYKLVVVGADGVGKSALTIQLIQ (KRAS(G12D)25mer_nt.STING(V155M)) 52MPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEELRHIHSRYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALNILLGLKGLAPAEISAVCEKGNFNMAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDHAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDRLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLRTDFSATNFSLLKQAGDVEENPGPMTEYKLVVVGAVGVGKSALTIQLIQ (KRAS(G12V)25mer_nt.STING(V155M) 53MPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEELRHIHSRYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALNILLGLKGLAPAEISAVCEKGNFNMAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDHAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDRLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLRTDFSATNFSLLKQAGDVEENPGPMTEYKLVVVGAGDVGKSALTIQLIQ (KRAS(G13D)25mer_nt.STING(V155M) 54MPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEELRHIHSRYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALNILLGLKGLAPAEISAVCEKGNFNMAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDHAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDRLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLRTDFSATNFSLLKQAGDVEENPGPMKLVVVGADGVGKSALKLVVVGADGVGKSALKLVVVGADGVGKSAL(KRAS(G12D)15mer^3_nt.STING(V155M)) 55MPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEELRHIHSRYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALNILLGLKGLAPAEISAVCEKGNFNMAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDHAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDRLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLRTDFSATNFSLLKQAGDVEENPGPMKLVVVGAVGVGKSALKLVVVGAVGVGKSALKLVVVGAVGVGKSAL(KRAS(G12V)15mer^3_nt.STING(V155M) 56MPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEELRHIHSRYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALNILLGLKGLAPAEISAVCEKGNFNMAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDHAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDRLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLRTDFSATNFSLLKQAGDVEENPGPMLVVVGAGDVGKSALTLVVVGAGDVGKSALTLVVVGAGDVGKSALT(KRAS(G13D)15mer^3_nt.STING(V155M) 57MPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEELRHIHSRYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALNILLGLKGLAPAEISAVCEKGNFNMAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDHAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDRLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLRTDFSATNFSLLKQAGDVEENPGPMTEYKLVVVGADGVGKSALTIQLIQMTEYKLVVVGADGVGKSALTIQLIQMTEYKLVVVGADGVGKSALTIQLIQ(KRAS(G12D)25mer^3_nt.STING(V155M)) 58MPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEELRHIHSRYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALNILLGLKGLAPAEISAVCEKGNFNMAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDHAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDRLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLRTDFSATNFSLLKQAGDVEENPGPMTEYKLVVVGAVGVGKSALTIQLIQMTEYKLVVVGAVGVGKSALTIQLIQMTEYKLVVVGAVGVGKSALTIQLIQ(KRAS(G12V)25mer^3_nt.STING(V155M) 59MPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEELRHIHSRYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALNILLGLKGLAPAEISAVCEKGNFNMAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDHAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDRLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLRTDFSATNFSLLKQAGDVEENPGPMTEYKLVVVGAGDVGKSALTIQLIQMTEYKLVVVGAGDVGKSALTIQLIQMTEYKLVVVGAGDVGKSALTIQLIQ(KRAS(G13D)25mer^3_nt.STING(V155M) 60MKLVVVGADGVGKSALATNFSLLKQAGDVEENPGPMPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEELRHIHSRYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALNILLGLKGLAPAEISAVCEKGNFNMAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDHAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDRLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLRTDFS (KRAS(G12D)15mer_ct.STING(V155M)) 61MKLVVVGAVGVGKSALATNFSLLKQAGDVEENPGPMPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEELRHIHSRYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALNILLGLKGLAPAEISAVCEKGNFNMAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDHAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDRLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLRTDFS (KRAS(G12V)15mer_ct.STING(V155M) 62MLVVVGAGDVGKSALTATNFSLLKQAGDVEENPGPMPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEELRHIHSRYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALNILLGLKGLAPAEISAVCEKGNFNMAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDHAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDRLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLRTDFS (KRAS(G13D)15mer_ct.STING(V155M) 63MTEYKLVVVGADGVGKSALTIQLIQATNFSLLKQAGDVEENPGPMPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEELRHIHSRYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALNILLGLKGLAPAEISAVCEKGNFNMAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDHAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDRLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLRTDFS (KRAS(G12D)25mer_ct.STING(V155M))64MTEYKLVVVGAVGVGKSALTIQLIQATNFSLLKQAGDVEENPGPMPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEELRHIHSRYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALNILLGLKGLAPAEISAVCEKGNFNMAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDHAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDRLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLRTDFS (KRAS(G12V)25mer_ct.STING(V155M)65MTEYKLVVVGAGDVGKSALTIQLIQATNFSLLKQAGDVEENPGPMPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEELRHIHSRYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALNILLGLKGLAPAEISAVCEKGNFNMAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDHAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDRLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLRTDFS (KRAS(G13D)25mer_ct.STING(V155M)66MKLVVVGADGVGKSALKLVVVGADGVGKSALKLVVVGADGVGKSALATNFSLLKQAGDVEENPGPMPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEELRHIHSRYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALNILLGLKGLAPAEISAVCEKGNFNMAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDHAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDRLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLRTDFS(KRAS(G12D)15mer^3_ct.STING(V155M)) 67MKLVVVGAVGVGKSALKLVVVGAVGVGKSALKLVVVGAVGVGKSALATNFSLLKQAGDVEENPGPMPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEELRHIHSRYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALNILLGLKGLAPAEISAVCEKGNFNMAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDHAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDRLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLRTDFS(KRAS(G12V)15mer^3_ct.STING(V155M) 68MLVVVGAGDVGKSALTLVVVGAGDVGKSALTLVVVGAGDVGKSALTATNFSLLKQAGDVEENPGPMPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEELRHIHSRYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALNILLGLKGLAPAEISAVCEKGNFNMAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDHAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDRLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLRTDFS(KRAS(G13D)15mer^3_ct.STING(V155M) 69MTEYKLVVVGADGVGKSALTIQLIQMTEYKLVVVGADGVGKSALTIQLIQMTEYKLVVVGADGVGKSALTIQLIQATNFSLLKQAGDVEENPGPMPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEELRHIHSRYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALNILLGLKGLAPAEISAVCEKGNFNMAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDHAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDRLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLRTDFS(KRAS(G12D)25mer^3_ct.STING(V155M)) 70MTEYKLVVVGAVGVGKSALTIQLIQMTEYKLVVVGAVGVGKSALTIQLIQMTEYKLVVVGAVGVGKSALTIQLIQATNFSLLKQAGDVEENPGPMPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEELRHIHSRYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALNILLGLKGLAPAEISAVCEKGNFNMAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDHAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDRLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLRTDFS(KRAS(G12V)25mer^3_ct.STING(V155M) 71MTEYKLVVVGAGDVGKSALTIQLIQMTEYKLVVVGAGDVGKSALTIQLIQMTEYKLVVVGAGDVGKSALTIQLIQATNFSLLKQAGDVEENPGPMPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEELRHIHSRYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALNILLGLKGLAPAEISAVCEKGNFNMAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDHAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDRLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLRTDFS(KRAS(G13D)25mer^3_ct.STING(V155M) 72 MTEYKLVVVGACGVGKSALTIQLIQ(KRAS(G12C)25mer) 73MTEYKLVVVGACGVGKSALTIQLIQMTEYKLVVVGACGVGKSALTIQLIQMTEYKLVVVGACGVGKSALTIQLIQ(KRAS(G12C)25mer^3) 74 MTEYKLVVVGAGGVGKSALTIQLIQ (KRAS(WT)25mer) 75MSAGDPRVGSGSLDSFMFSIPLVALNVGVRRRLSLFLNPRTPVAADWTLLAEEMGFEYLEIRELETRPDPTRSLLDAWQGRSGASVGRLLELLALLDREDILKELKSRIEEDCQKYLGKQQNQESEKPLQVARVESSVPQTKELGGITTLDDPLGQTPELFDAFICYCPNDIEFVQEMIRQLEQTDYRLKLCVSDRDVLPGTCVWSIASELIEKRCRRMVVVVSDDYLQSKECDFQTKFALSLSPGVQQKRPIPIKYKAMKKDFPSILRFITICDYTNPCTKSWFWTRLAKALSLP (humanmyd88(L265P); P4027 without epitope tag) 76MAAGGPGAGSAAPVSSTSSLPLAALNMRVRRRLSLFLNVRTQVAADWTALAEEMDFEYLEIRQLETQADPTGRLLDAWQGRPGASVGRLLELLTKLGRDDVLLELGPSIEEDCQKYILKQQQEEAEKPLQVAAVDSSVPRTAELAGITTLDDPLGHMPERFDAFICYCPSDIQFVQEMIRQLEQTNYRLKLCVSDRDVLPGTCVWSIASELIEKRCRRMVVVVSDDYLQSKECDFQTKFALSLSPGAHQKRPIPIKYKAMKKEFPSILRFITVCDYTNPCTKSWFWTRLAKALSLP (mousemyd88(L265P); P4028 without epitope tag) 77MGVGKSKLDKCPLSWHKKDSVDADQDGHESDSKNSEEACLRGFVEQSSGSEPPTGEQDQPEAKGAGPEEQDEEEFLKFVILHAEDDTDEALRVQDLLQNDFGIRPGIVFAEMPCGRLHLQNLDDAVNGSAWTILLLTENFLRDTWCNFQFYTSLMNSVSRQHKYNSVIPMRPLNSPLPRERTPLALQTINALEEESQGFSTQVERIFRESVFERQQSIWKETRSVSQKQFIA(Mouse TRAM (TICAM2); P4033 without epitope tag) 78MSLWGLVSKMPPEKVQRLYVDFPQHLRHLLGDWLESQPWEFLVGSDAFCCNLASALLSDTVQHLQASVGEQGEGSTILQHISTLESIYQRDPLKLVATFRQILQGEKKAVMEQFRHLPMPFHWKQEELKFKTGLRRLQHRVGEIHLLREALQKGAEAGQVSLHSLIETPANGTGPSEALAMLLQETTGELEAAKALVLKRIQIWKRQQQLAGNGAPFEESLAPLQERCESLVDIYSQLQQEVGAAGGELEPKTRASLTGRLDEVLRTLVTSCFLVEKQPPQVLKTQTKFQAGVRFLLGLRFLGAPAKPPLVRADMVTEKQARELSVPQGPGAGAESTGEIINNTVPLENSIPGNCCSALFKNLLLKKIKRCERKGTESVTEEKCAVLFSASFTLGPGKLPIQLQALSLPLVVIVHGNQDNNAKATILWDNAFSEMDRVPFVVAERVPWEKMCETLNLKFMAEVGTNRGLLPEHFLFLAQKIFNDNSLSMEAFQHRSVSWSQFNKEILLGRGFTFWQWFDGVLDLTKRCLRSYWSDRLIIGFISKQYAASLLLNEPDGTFLLRFSDSEIGGITIAHVIRGQDGSPQIENIQPFSAKDLSIRSLGDRIRDLAQLKNLYPKKPKDEAFRSHYKPEQMGKDGRGYVPATIKMTVERDQPLPTPELQMPTMVPSYDLGMAPDSSMSMQLGPDMVPQVYPPHSHSIPPYQGLSPEESVNVLSAFQEPHLQMPPSLGQMSLPFDQPHPQGLLPCQPQEHAVSSPDPLLCSDVTMVEDSCLSQPVTAFPQGTWIGEDIFPPLLPPTEQDLTKLLLEGQGESGGGSLGAQPLLQPSHYGQSGISMSHMDLRANPSW(STAT6 V547A/T548A); P008 with no epitope tag) 79MSLWGLVSKMPPEKVQRLYVDFPQHLRHLLGDWLESQPWEFLVGSDAFCCNLASALLSDTVQHLQASVGEQGEGSTILQHISTLESIYQRDPLKLVATFRQILQGEKKAVMEQFRHLPMPFHWKQEELKFKTGLRRLQHRVGEIHLLREALQKGAEAGQVSLHSLIETPANGTGPSEALAMLLQETTGELEAAKALVLKRIQIWKRQQQLAGNGAPFEESLAPLQERCESLVDIYSQLQQEVGAAGGELEPKTRASLTGRLDEVLRTLVTSCFLVEKQPPQVLKTQTKFQAGVRFLLGLRFLGAPAKPPLVRADMVTEKQARELSVPQGPGAGAESTGEIINNTVPLENSIPGNCCSALFKNLLLKKIKRCERKGTESVTEEKCAVLFSASFTLGPGKLPIQLQALDLPLVVIVHGNQDNNAKATILWDNAFSEMDRVPFVVAERVPWEKMCETLNLKFMAEVGTNRGLLPEHFLFLAQKIFNDNSLSMEAFQHRSVSWSQFNKEILLGRGFTFWQWFDGVLDLTKRCLRSYWSDRLIIGFISKQYVTSLLLNEPDGTFLLRFSDSEIGGITIAHVIRGQDGSPQIENIQPFSAKDLSIRSLGDRIRDLAQLKNLYPKKPKDEAFRSHYKPEQMGKDGRGYVPATIKMTVERDQPLPTPELQMPTMVPSYDLGMAPDSSMSMQLGPDMVPQVYPPHSHSIPPYQGLSPEESVNVLSAFQEPHLQMPPSLGQMSLPFDQPHPQGLLPCQPQEHAVSSPDPLLCSDVTMVEDSCLSQPVTAFPQGTWIGEDIFPPLLPPTEQDLTKLLLEGQGESGGGSLGAQPLLQPSHYGQSGISMSHMDLRANPSW(STAT6 (S407D); P009 with no epitope tag) 80MSLWGLVSKMPPEKVQRLYVDFPQHLRHLLGDWLESQPWEFLVGSDAFCCNLASALLSDTVQHLQASVGEQGEGSTILQHISTLESIYQRDPLKLVATFRQILQGEKKAVMEQFRHLPMPFHWKQEELKFKTGLRRLQHRVGEIHLLREALQKGAEAGQVSLHSLIETPANGTGPSEALAMLLQETTGELEAAKALVLKRIQIWKRQQQLAGNGAPFEESLAPLQERCESLVDIYSQLQQEVGAAGGELEPKTRASLTGRLDEVLRTLVTSCFLVEKQPPQVLKTQTKFQAGVRFLLGLRFLGAPAKPPLVRADMVTEKQARELSVPQGPGAGAESTGEIINNTVPLENSIPGNCCSALFKNLLLKKIKRCERKGTESVTEEKCAVLFSASFTLGPGKLPIQLQALDLPLVVIVHGNQDNNAKATILWDNAFSEMDRVPFVVAERVPWEKMCETLNLKFMAEVGTNRGLLPEHFLFLAQKIFNDNSLSMEAFQHRSVSWSQFNKEILLGRGFTFWQWFDGVLDLTKRCLRSYWSDRLIIGFISKQYAASLLLNEPDGTFLLRFSDSEIGGITIAHVIRGQDGSPQIENIQPFSAKDLSIRSLGDRIRDLAQLKNLYPKKPKDEAFRSHYKPEQMGKDGRGYVPATIKMTVERDQPLPTPELQMPTMVPSYDLGMAPDSSMSMQLGPDMVPQVYPPHSHSIPPYQGLSPEESVNVLSAFQEPHLQMPPSLGQMSLPFDQPHPQGLLPCQPQEHAVSSPDPLLCSDVTMVEDSCLSQPVTAFPQGTWIGEDIFPPLLPPTEQDLTKLLLEGQGESGGGSLGAQPLLQPSHYGQSGISMSHMDLRANPSW(STAT6 (S407D/V547A/T548A); P010 with no epitope tag) 81MSLWGLVSKMPPEKVQRLYVDFPQHLRHLLGDWLESQPWEFLVGSDAFCCNLASALLSDTVQHLQASVGEQGEGSTILQHISTLESIYQRDPLKLVATFRQILQGEKKAVMEQFRHLPMPFHWKQEELKFKTGLRRLQHRVGEIHLLREALQKGAEAGQVSLHSLIETPANGTGPSEALAMLLQETTGELEAAKALVLKRIQIWKRQQQLAGNGAPFEESLAPLQERCESLVDIYSQLQQEVGAAGGELEPKTRASLTGRLDEVLRTLVTSCFLVEKQPPQVLKTQTKFQAGVRFLLGLRFLGAPAKPPLVRADMVTEKQARELSVPQGPGAGAESTGEIINNTVPLENSIPGNCCSALFKNLLLKKIKRCERKGTESVTEEKCAVLFSASFTLGPGKLPIQLQALSLPLVVIVHGNQDNNAKATILWDNAFSEMDRVPFVVAERVPWEKMCETLNLKFMAEVGTNRGLLPEHFLFLAQKIFNDNSLSMEAFQHRSVSWSQFNKEILLGRGFTFWQWFDGVLDLTKRCLRSYWSDRLIIGFISKQYAASLLLNEPDGTFLLRFSDSEIGGITIAHVIRGQDGSPQIENIQPFSAKDLSIRSLGDRIRDLAQLKNLYPKKPKDEAFRSHYKPEQMGKDGRGFVPATIKMTVERDQPLPTPELQMPTMVPSYDLGMAPDSSMSMQLGPDMVPQVYPPHSHSIPPYQGLSPEESVNVLSAFQEPHLQMPPSLGQMSLPFDQPHPQGLLPCQPQEHAVSSPDPLLCSDVTMVEDSCLSQPVTAFPQGTWIGEDIFPPLLPPTEQDLTKLLLEGQGESGGGSLGAQPLLQPSHYGQSGISMSHMDLRANPSW(STAT6 (V547A/T548A/Y641F); P011 with no epitope tag) 82SAEVIHQVEEALDTDEKEMLLFLCRDVAIDVVPPNVRDLLDILRERGKLSVGDLAELLYRVRRFDLLKRILKMDRKAVETHLLRNPHLVSDYRVLMAEIGEDLDKSDVSSLIFLMKDYMGRGKISKEKSFLDLVVELEKLNLVAPDQLDLLEKCLKNIHRIDLKTKIQKYKQSVQGAGTSYRNVLQAAIQKSLKDPSNNFRLHNGRSKEQRLKEQLGAQQEPVKKSIQESEAFLPQSIPEERYKMKSKPLGICLIIDCIGNETELLRDTFTSLGYEVQKFLHLSMHGISQILGQFACMPEHRDYDSFVCVLVSRGGSQSVYGVDQTHSGLPLHHIRRMFMGDSCPYLAGKPKMFFIQNYVVSEGQLEDSSLLEVDGPAMKNVEFKAQKRGLCTVHREADFFWSLCTADMSLLEQSHSSPSLYLQCLSQKLRQERKRPLLDLHIELNGYMYDWNSRVSAKEKYYVWLQHTLRKKLILSYT (hu-cFLIP-L; P1006 without epitope tag) 83SAEVIHQVEEALDTDEKEMLLFLCRDVAIDVVPPNVRDLLDILRERGKLSVGDLAELLYRVRRFDLLKRILKMDRKAVETHLLRNPHLVSDYRVLMAEIGEDLDKSDVSSLIFLMKDYMGRGKISKEKSFLDLVVELEKLNLVAPDQLDLLEKCLKNIHRIDLKTKIQKYKQSVQGAGTSYRNVLQAAIQKSLKDPSNNFRLHNGRSKEQRLKEQLGAQQEPVKKS(hu-cFLIP-S(1-227); P1007 without epitope tag) 84SAEVIHQVEEALDTDEKEMLLFLCRDVAIDVVPPNVRDLLDILRERGKLSVGDLAELLYRVRRFDLLKRILKMDRKAVETHLLRNPHLVSDYRVLMAEIGEDLDKSDVSSLIFLMKDYMGRGKISKEKSFLDLVVELEKLNLVAPDQLDLLEKCLKNIHRIDLKTKIQKYKQSVQGAGTSYRNVLQAAIQKSLKD (hu-cFLIP-p22(1-198); P1008 withoutepitope tag) 85SAEVIHQVEEALDTDEKEMLLFLCRDVAIDVVPPNVRDLLDILRERGKLSVGDLAELLYRVRRFDLLKRILKMDRKAVETHLLRNPHLVSDYRVLMAEIGEDLDKSDVSSLIFLMKDYMGRGKISKEKSFLDLVVELEKLNLVAPDQLDLLEKCLKNIHRIDLKTKIQKYKQSVQGAGTSYRNVLQAAIQKSLKDPSNNFRLHNGRSKEQRLKEQLGAQQEPVKKSIQESEAFLPQSIPEERYKMKSKPLGICLIIDCIGNETELLRDTFTSLGYEVQKFLHLSMHGISQILGQFACMPEHRDYDSFVCVLVSRGGSQSVYGVDQTHSGLPLHHIRRMFMGDSCPYLAGKPKMFFIQNYVVSEGQLEDSSLLEVD(hu-cFLIP-p43(1-376); P1009 without epitope tag) 86GPAMKNVEFKAQKRGLCTVHREADFFWSLCTADMSLLEQSHSSPSLYLQCLSQKLRQERKRPLLDLHIELNGYMYDWNSRVSAKEKYYVWLQHTLRKKLILSYT (hu-cFLIP-p12(377-480); P1010 withoutepitope tag) 87MSWSPSLTTQTCGAWEMKERLGTGGFGNVIRWHNQETGEQIAIKQCRQELSPRNRERWCLEIQIMRRLTHPNVVAARDVPEGMQNLAPNDLPLLAMEYCQGGDLRKYLNQFENCCGLREGAILTLLSDIASALRYLHENRIIHRDLKPENIVLQQGEQRLIHKIIDLGYAKELDQGELCTEFVGTLQYLAPELLEQQKYTVTVDYWSFGTLAFECITGFRPFLPNWQPVQWHSKVRQKSEVDIVVSEDLNGTVKFSSSLPYPNNLNSVLAERLEKWLQLMLMWHPRQRGTDPTYGPNGCFKALDDILNLKLVHILNMVTGTIHTYPVTEDESLQSLKARIQQDTGIPEEDQELLQEAGLALIPDKPATQCISDGKLNEGHTLDMDLVFLFDNSKITYETQISPRPQPESVSCILQEPKRNLAFFQLRKVWGQVWHSIQTLKEDCNRLQQGQRAAMMNLLRNNSCLSKMKNSMASMSQQLKAKLDFFKTSIQIDLEKYSEQTEFGITSDKLLLAWREMEQAVELCGRENEVKLLVERMMALQTDIVDLQRSPMGRKQGGTLDDLEEQARELYRRLREKPRDQRTEGDSQEMVRLLLQAIQSFEKKVRVIYTQLSKTVVCKQKALELLPKVEEVVSLMNEDEKTVVRLQEKRQKELWNLLKIACSKVRGPVSGSPDSMNASRLSQPGQLMSQPSTASNSLPEPAKKSEELVAEAHNLCTLLENAIQDTVREQDQSFTALDWSWLQTEEEEHSCLEQAS (hulKK2ca(S177E/S181E);P4005 without epitope tag) 88MSWSPSLTTQTCGAWEMKERLGTGGFGNVIRWHNQETGEQIAIKQCRQELSPRNRERWCLEIQIMRRLTHPNVVAARDVPEGMQNLAPNDLPLLAMEYCQGGDLRKYLNQFENCCGLREGAILTLLSDIASALRYLHENRIIHRDLKPENIVLQQGEQRLIHKIIDLGYAKELDQGALCTAFVGTLQYLAPELLEQQKYTVTVDYWSFGTLAFECITGFRPFLPNWQPVQWHSKVRQKSEVDIVVSEDLNGTVKFSSSLPYPNNLNSVLAERLEKWLQLMLMWHPRQRGTDPTYGPNGCFKALDDILNLKLVHILNMVTGTIHTYPVTEDESLQSLKARIQQDTGIPEEDQELLQEAGLALIPDKPATQCISDGKLNEGHTLDMDLVFLFDNSKITYETQISPRPQPESVSCILQEPKRNLAFFQLRKVWGQVWHSIQTLKEDCNRLQQGQRAAMMNLLRNNSCLSKMKNSMASMSQQLKAKLDFFKTSIQIDLEKYSEQTEFGITSDKLLLAWREMEQAVELCGRENEVKLLVERMMALQTDIVDLQRSPMGRKQGGTLDDLEEQARELYRRLREKPRDQRTEGDSQEMVRLLLQAIQSFEKKVRVIYTQLSKTVVCKQKALELLPKVEEVVSLMNEDEKTVVRLQEKRQKELWNLLKIACSKVRGPVSGSPDSMNASRLSQPGQLMSQPSTASNSLPEPAKKSEELVAEAHNLCTLLENAIQDTVREQDQSFTALDWSWLQTEEEEHSCLEQAS(hulKK2null(S177A/S181A); P4006 without epitope tag) 89MSWSPSLPTQTCGAWEMKERLGTGGFGNVIRWHNQATGEQIAIKQCRQELSPKNRNRWCLEIQIMRRLNHPNVVAARDVPEGMQNLAPNDLPLLAMEYCQGGDLRRYLNQFENCCGLREGAVLTLLSDIASALRYLHENRIIHRDLKPENIVLQQGEKRLIHKIIDLGYAKELDQGELCTEFVGTLQYLAPELLEQQKYTVTVDYWSFGTLAFECITGFRPFLPNWQPVQWHSKVRQKSEVDIVVSEDLNGAVKFSSSLPFPNNLNSVLAERLEKWLQLMLMWHPRQRGTDPQYGPNGCFRALDDILNLKLVHVLNMVTGTVHTYPVTEDESLQSLKTRIQENTGILETDQELLQKAGLVLLPDKPATQCISDSKTNEGLTLDMDLVFLLDNSKINYETQITPRPPPESVSCILQEPKRNLSFFQLRKVWGQVWHSIQTLKEDCNRLQQGQRAAMMSLLRNNSCLSKMKNAMASTAQQLKAKLDFFKTSIQIDLEKYKEQTEFGITSDKLLLAWREMEQAVEQCGRENDVKHLVERMMALQTDIVDLQRSPMGRKQGGTLDDLEEQARELYRKLREKPRDQRTEGDSQEMVRLLLQAIQSFEKKVRVIYTQLSKTVVCKQKALELLPKVEEVVSLMNEDERTVVRLQEKRQKELWNLLKIACSKVRGPVSGSPDSMNVSRLSHPGQLMSQPSSACDSLPESDKKSEELVAEAHALCSRLESALQDTVKEQDRSFTTLDWSWLQMEDEERCSLEQACD (mulKK2ca(S177E/S181E); P4002 without epitope tag) 90MSWSPSLPTQTCGAWEMKERLGTGGFGNVIRWHNQATGEQIAIKQCRQELSPKNRNRWCLEIQIMRRLNHPNVVAARDVPEGMQNLAPNDLPLLAMEYCQGGDLRRYLNQFENCCGLREGAVLTLLSDIASALRYLHENRIIHRDLKPENIVLQQGEKRLIHKIIDLGYAKELDQGALCTAFVGTLQYLAPELLEQQKYTVTVDYWSFGTLAFECITGFRPFLPNWQPVQWHSKVRQKSEVDIVVSEDLNGAVKFSSSLPFPNNLNSVLAERLEKWLQLMLMWHPRQRGTDPQYGPNGCFRALDDILNLKLVHVLNMVTGTVHTYPVTEDESLQSLKTRIQENTGILETDQELLQKAGLVLLPDKPATQCISDSKTNEGLTLDMDLVFLLDNSKINYETQITPRPPPESVSCILQEPKRNLSFFQLRKVWGQVWHSIQTLKEDCNRLQQGQRAAMMSLLRNNSCLSKMKNAMASTAQQLKAKLDFFKTSIQIDLEKYKEQTEFGITSDKLLLAWREMEQAVEQCGRENDVKHLVERMMALQTDIVDLQRSPMGRKQGGTLDDLEEQARELYRKLREKPRDQRTEGDSQEMVRLLLQAIQSFEKKVRVIYTQLSKTVVCKQKALELLPKVEEVVSLMNEDERTVVRLQEKRQKELWNLLKIACSKVRGPVSGSPDSMNVSRLSHPGQLMSQPSSACDSLPESDKKSEELVAEAHALCSRLESALQDTVKEQDRSFTTLDWSWLQMEDEERCSLEQACD mulKK2null(S177A/S181A); P4003 without epitope tag) 91MERPPGLRPGAGGPWEMRERLGTGGFGNVCLYQHRELDLKIAIKSCRLELSTKNRERWCHEIQIMKKLNHANVVKACDVPEELNILIHDVPLLAMEYCSGGDLRKLLNKPENCCGLKESQILSLLSDIGSGIRYLHENKIIHRDLKPENIVLQDVGGKIIHKIIDLGYAKDVDQGELCTEFVGTLQYLAPELFENKPYTATVDYWSFGTMVFECIAGYRPFLHHLQPFTWHEKIKKKDPKCIFACEEMSGEVRFSSHLPQPNSLCSLVVEPMENWLQLMLNWDPQQRGGPVDLTLKQPRCFVLMDHILNLKIVHILNMTSAKIISFLLPPDESLHSLQSRIERETGINTGSQELLSETGISLDPRKPASQCVLDGVRGCDSYMVYLFDKSKTVYEGPFASRSLSDCVNYIVQDSKIQLPIIQLRKVWAEAVHYVSGLKEDYSRLFQGQRAAMLSLLRYNANLTKMKNTLISASQQLKAKLEFFHKSIQLDLERYSEQMTYGISSEKMLKAWKEMEEKAIHYAEVGVIGYLEDQIMSLHAEIMELQKSPYRRQGDLMESLEQRAIDLYKQLKHRPSDHSYSDSTEMVKIIVHTVQSQDRVLKELFGHLSKLLGCKQKIIDLLPKVEVALSNIKEADNTVMFMQGKRQKEIWHLLKIACTQAAARALVGAALEGAVAPQAAAWLPPAAAEHDHALACVVAPQDGEAAAQMIEENLNCLGHLAAIIHEANEEQGNSMMNLDWSWLTE Human constitutively active IKKalpha (PEST mutation) P.4013 without epitope tag 92MERPPGLRPGAGGPWEMRERLGTGGFGNVCLYQHRELDLKIAIKSCRLELSTKNRERWCHEIQIMKKLNHANVVKACDVPEELNILIHDVPLLAMEYCSGGDLRKLLNKPENCCGLKESQILSLLSDIGSGIRYLHENKIIHRDLKPENIVLQDVGGKIIHKIIDLGYAKDVDQGELCTEFVGTLQYLAPELFENKPYTATVDYWSFGTMVFECIAGYRPFLHHLQPFTWHEKIKKKDPKCIFACEEMSGEVRFSSHLPQPNSLCSLVVEPMENWLQLMLNWDPQQRGGVDLTLKQPRCFVLMDHILNLKIVHILNMTSAKIISFLLPPDESLHSLQSRIERETGINTGSQELLSETGISLDPRKPASQCVLDGVRGCDSYMVYLFDKSKTVYEGPFASRSLSDCVNYIVQDSKIQLPIIQLRKVWAEAVHYVSGLKEDYSRLFQGQRAAMLSLLRYNANLTKMKNTLISASQQLKAKLEFFHKSIQLDLERYSEQMTYGISSEKMLKAWKEMEEKAIHYAEVGVIGYLEDQIMSLHAEIMELQKSPYRRQGDLMESLEQRAIDLYKQLKHRPSDHSYSDSTEMVKIIVHTVQSQDRVLKELFGHLSKLLGCKQKIIDLLPKVEVALSNIKEADNTVMFMQGKRQKEIWHLLKIACTQAAARALVGAALEGAVAPQAAAWLPPAAAEHDHALACVVAPQDGEAAAQMIEENLNCLGHLAAIIHEANEEQGNSMMNLDWSWLTE Human constitutively active IKKalpha (PEST mutation) P.4014 without epitope tag 93MSWSPSLTTQTCGAWEMKERLGTGGFGNVIRWHNQETGEQIAIKQCRQELSPRNRERWCLEIQIMRRLTHPNVVAARDVPEGMQNLAPNDLPLLAMEYCQGGDLRKYLNQFENCCGLREGAILTIISDIASALRYLHENRIIHRDLKPENIVLQQGEQRLIHKIIDLGYAKELDQGELCTEFVGTLQYLAPELLEQQKYVTVDYWSFGTLAFECITGFRPFLPNWQPVQWHSKVRQKSEVDIVVSEDLNGTVKFSSSLPYPNNLNSVLAERLEKWLQLMLMWHPRQRGTDPTYGPNGCFKALDDILNLKLVHILNMVTGTIHTYPVTEDESLQSLKARIQQDTGIPEEDQELLQEAGLALIPDKPATQCISDGKLNEGHTLDMDLVFLFDNISKITYETQISPRPQPESVSCILQEPKRNLAFFQLRKVWGQVWHSIQTLKEDCNRLQQGQRAAMMNLLRNNSCLSKMKNSMASMSQQLKAKLDFFKTISIQIDLEKYSEQTEFGITSDKLLLAWREMEQAVELCGRENEVKLLVERMMALQTDIVDLQRSPMGRKQGGTLDDLEEQARELYRRLREKPRDQRTEGDSQEMVRLLLQAIQSFEKKVRVIYTQLSKTVVCKQKALELLPKVEEVVSLMNEDEKTVVRLQEKRQKELWNLLKIACSKVRGPVAGAPDAMNAARLAQPGQLMAQPATAANALPEPAKKAEELVAEAHNLCTLLENAIQDTVREQDQSFTALDWSWLQTEEEEHSCLEQAS Humanconstitutively active IKK beta (PEST mutation) P.4015 without epitopetag 94MSWSPSLTTQTCGAWEMKERLGTGGFGNVIRWHNQETGEQIAIKQCRQELSPRNRERWCLEIQIMRRLTHPNVVAARDVPEGMQNLAPNDLPLLAMEYCQGGDLRKYLNQFENCCGLREGAILTLLSDIASALRYLHENRIIHRDLKPENIVLQQGEQRLIHKIIDLGYAKELDQGELCTEFVGTLQYLAPELLEQQKYTVTVDYWSFGTLAFECITGFRPFLPNWQPVQWHSKVRQKSEVDIVVSEDLNGTVKFSSSLPYPNNLNSVLAERLEKWLQLMLMWHPRQRGTDPTYGPNGCFKALDDILNLKLVHILNMVTGTIHTYPVTEDESLQSLKARIQQDTGIPEEDQELLQEAGLALIPDKPATQCISDGKLNEGHTLDMDLVFLFDNSKITYETQISPRPQPESVSCILQEPKRNLAFFQLRKVWGQVWHSIQTLKEDCNRLQQGQRAAMMNLLRNNSCLSKMKNSMASMSQQLKAKLDFFKTSIQIDLEKYSEQTEFGITSDKLLLAWREMEQAVELCGRENEVKLLVERMMALQTDIVDLQRSPMGRKQGGTLDDLEEQARELYRRLREKPRDQRTEGDSQEMVRLLLQAIQSFEKKVRVIYTQLSKTVVCKQKALELLPKVEEVVSLMNEDEKTVVRLQEKRQKELWNLLKIACSKVRGPVAGAPDAMNARRLAQPGQLMAQPATAANALPEPAKKAEELVAEAHNLCTLLENAIQDTVREQDQSFTALDWSWLQTEEEEHSCLEQAS Humanconstitutively active IKK beta (PEST mutation) P.4016 without epitopetag 95MERPPGLRPGAGGPWEMRERLGTGGFGNVSLYQHRELDLKIAIKSCRLELSSKNRERWCHEIQIMKKLDHANVVKACDVPEELNFLINDVPLLAMEYCSGGDLRKLLNKPENCCGLKESQILSLLSDIGSGIRYLHENKIIHRDLKPENIVLQDVGGKTIHKIIDLGYAKDVDQGELCTEFVGTLQYLAPELFENKPYTATVDYWSFGTMVFECIAGYRPFLHHLQPFTWHEKIKKKDPKCIFACEEMTGEVRFSSHLPQPNSLCSLIVEPMESWLQLMLNWDPQQRGGPIDLTLKQPRCFALMDHILNLKIVHILNMTSAKIISFLLPCDESLHSLQSRIERETGINTGSQELLSETGISLDPRKPASQCVLDGVRGCDSYMVYLFDKSKTVYEGPFASRSLSDCVNYIVQDSKIQLPIIQLRKVWAEAVHYVSGLKEDYSRLFQGQRAAMLSLLRYNANLTKMKNTLISASQQLKAKLEFFRKSIQLDLERYSEQMTYGISSEKMLKAWKEMEEKAIHYSEVGVIGYLEDQIMSLHTEIMELQKSPYGRRQGDLMESLEQRAIDLYKQLKHRPPDHLYSDSTEMVKIIVHTVQSQDRVLKELFGHLSKLLGCKQKIIDLLPKVEVALSNIKEADNTVMFMQGKRQKEIWHLLKIACTQAAARALVGAALEGAVAPPVAAWLPPALADREHPLTCVVAPQDGEALAQMIEENLNCLGHLAAIIREANEDQSSSLMSLDWSWLAE Mouse constitutively active IKKalpha (PEST mutation) P.4017 without epitope tag 96MERPPGLRPGAGGPWEMRERLGTGGFGNVSLYQHRELDLKIAIKSCRLELSSKNRERWCHEIQIMKKLDHANVVKACDVPEELNFLINDVPLLAMEYCSGGDLRKLLNKPENCCGLKESQILSLLSDIGSGIRYLHENKIIHRDLKPENIVLQDVGGKTIHKIIDLGYAKDVDQGELCTEFVGTLQYLAPELFENKPYTATVDYWSFGTMVFECIAGYRPFLHHLQPFTWHEKIKKKDPKCIFACEEMTGEVRFSSHLPQPNSLCSLIVEPMESWLQLMLNWDPQQRGGPIDLTLKQPRCFALMDHILNLKIVHILNMTSAKIISFLLPCDESLHSLQSRIERETGINTGSQELLSETGISLDPRKPASQCVLDGVRGCDSYMVYLFDKSKTVYEGPFASRSLSDCVNYIVQDSKIQLPIIQLRKVWAEAVHYVSGLKEDYSRLFQGQRAAMLSLLRYNANLTKMKNTLISASQQLKAKLEFFRKSIQLDLERYSEQMTYGISSEKMLKAWKEMEEKAIHYSEVGVIGYLEDQIMSLHTEIMELQKSPYGRRQGDLMESLEQRAIDLYKQLKHRPPDHLYSDSTEMVKIIVHTVQSQDRVLKELFGHLSKLLGCKQKIIDLLPKVEVALSNIKEADNTVMFMQGKRQKEIWHLLKIACTQAAARALVGAALEGAVAPPVAAWLPPALADREHPLTCVVAPQDGEALAQMIEENLNCLGHLAAIIREANEDQSSSLMSLDWSWLAE Mouse constitutively active IKKalpha (PEST mutation) P.4018 without epitope tag 97MSWSPSLPTQTCGAWEMKERLGTGGFGNVIRWHNQATGEQIAIKQCRQELSPKNRNRWCLEIQIMRRLNHPNVVAARDVPEGMQNLAPNDLPLLAMEYCQGGDLRRYLNQFENCCGLREGAVLTLLSDIASALRYLHENRIIHRDLKPENIVLQQGEKRLIHKIIDLGYAKELDQGELCTEFVGTLQYLAPELLEQQKYTVTVDYWSFGTLAFECITGFRPFLPNWQPVQWHSKVRQKSEVDIVVSEDLNGAVKFSSSLPFPNNLNSVLAERLEKWLQLMLMWHPRQRGTDPQYGPNGCFRALDDILNLKLVHVLNMVTGTVHTYPVTEDESLQSLKTRIQENTGILETDQELLQKAGLVLLPDKPATQCISDSKTNEGLTLDMDLVFLLDNSKINYETQITPRPPPESVSCILQEPKRNLSFFQLRKVWGQVWHSIQTLKEDCNRLQQGQRAAMMSLLRNNSCLSKMKNAMASTAQQLKAKLDFFKTSIQIDLEKYKEQTEFGITSDKLLLAWREMEQAVEQCGRENDVKHLVERMMALQTDIVDLQRSPMGRKQGGTLDDLEEQARELYRKLREKPRDQRTEGDSQEMVRLLLQAIQSFEKKVRVIYTQLSKTVVCKQKALELLPKVEEVVSLMNEDERTVVRLQEKRQKELWNLLKIACSKVRGPVAGAPDAMNVARLAHPGQLMAQPASACDALPESDKKAEELVAEAHALCSRLESALQDTVKEQDRSFTTLDWSWLQMEDEERCSLEQACDMouse constitutively active IKK beta (PEST mutation) P.4019 withoutepitope tag 98MSWSPSLPTQTCGAWEMKERLGTGGFGNVIRWHNQATGEQIAIKQCRQELSPKNRNRWCLEIQIMRRLNHPNVVAARDVPEGMQNLAPNDLPLLAMEYCQGGDLRRYLNQFENCCGLREGAVLTLLSDIASALRYLHENRIIHRDLKPENIVLQQGEKRLIHKIIDLGYAKELDQGELCTEFVGTLQYLAPELLEQQKYTVTVDYWSFGTLAFECITGFRPFLPNWQPVQWHSKVRQKSEVDIVVSEDLNGAVKFSSSLPFPNNLNSVLAERLEKWLQLMLMWHPRQRGTDPQYGPNGCFRALDDILNLKLVHVLNMVTGTVHTYPVTEDESLQSLKTRIQENTGILETDQELLQKAGLVLLPDKPATQCISDSKTNEGLTLDMDLVFLLDNSKINYETQITPRPPPESVSCILQEPKRNLSFFQLRKVWGQVWHSIQTLKEDCNRLQQGQRAAMMSLLRNNSCLSKMKNAMASTAQQLKAKLDFFKTSIQIDLEKYKEQTEFGITSDKLLLAWREMEQAVEQCGRENDVKHLVERMMALQTDIVDLQRSPMGRKQGGTLDDLEEQARELYRKLREKPRDQRTEGDSQEMVRLLLQAIQSFEKKVRVIYTQLSKTVVCKQKALELLPKVEEVVSLMNEDERTVVRLQEKRQKELWNLLKIACSKVRGPVAGAPDAMNVARLAHPGQLMAQPASACDALPESDKKAEELVAEAHALCSRLESALQDTVKEQDRSFTTLDWSWLQMEDEERCSLEQACDMouse constitutively active IKK beta (PEST mutation) P.4020 withoutepitope tag 99MQPDMSLNVIKMKSSDFLESAELDSGGFGKVSLCFHRTQGLMIMKTVYKGPNCIEHNEALLEEAKMMNRLRHSRVVKLLGVIIEEGKYSLVMEYMEKGNLMHVLKAEMSTPLSVKGRIILEIIEGMCYLHGKGVIHKDLKPENILVDNDFHIKIADLGLASFKMWSKLNNEEHNELREVDGTAKKNGGTLYYMAPEHLNDVNAKPTEKSDVYSFAVVLWAIFANKEPYENAICEQQLIMCIKSGNRPDVDDITEYCPREIISLMKLCWEANPEARPTFPGIEEKFRPFYLSQLEESVEEDVKSLKKEYSNENAVVKRMQSLQLDCVAVPSSRSNSATEQPGSLHSSQGLGMGPVEESWFAPSLEHPQEENEPSLQSKLQDEANYHLYGSRMDRQTKQQPRQNVAYNREEERRRRVSHDPFAQQRPYENFQNTEGKGTAYSSAASHGNAVHQPSGLTSQPQVLYQNNGLYSSHGFGTRPLDPGTAGPRVWYRPIPSHMPSLHNIPVPETNYLGNTPTMPFSSLPPTDESIKYTIYNSTGIQIGAYNYMEIGGTSSSGGIKKEIEAIKKEQEAIKKKIEAIEKEIEA (huRIPK1(1-555).IZ.TM; TH1021without epitope tag) 100MQPDMSLNVIKMKSSDFLESAELDSGGFGKVSLCFHRTQGLMIMKTVYKGPNCIEHNEALLEEAKMMNRLRHSRVVKLLGVIIEEGKYSLVMEYMEKGNLMHVLKAEMSTPLSVKGRIILEIIEGMCYLHGKGVIHKDLKPENILVDNDFHIKIADLGLASFKMWSKLNNEEHNELREVDGTAKKNGGTLYYMAPEHLNDVNAKPTEKSDVYSFAVVLWAIFANKEPYENAICEQQLIMCIKSGNRPDVDDITEYCPREIISLMKLCWEANPEARPTFPGIEEKFRPFYLSQLEESVEEDVKSLKKEYSNENAVVKRMQSLQLDCVAVPSSRSNSATEQPGSLHSSQGLGMGPVEESWFAPSLEHPQEENEPSLQSKLQDEANYHLYGSRMDRQTKQQPRQNVAYNREEERRRRVSHDPFAQQRPYENFQNTEGKGTAYSSAASHGNAVHQPSGLTSQPQVLYQNNGLYSSHGFGTRPLDPGTAGPRVWYRPIPSHMPSLHNIPVPETNYLGNTPTMPFSSLPPTDESIKYTIYNSTGIQIGAYNYMEIGGTSSSGSDGSGSGSGSITIRAAFLEKENTALRTEIAELEKEVGRCENIVSKYETRYGPL(huRIPK1(1-555).EE.DM; TH1022 without epitope tag) 101MQPDMSLNVIKMKSSDFLESAELDSGGFGKVSLCFHRTQGLMIMKTVYKGPNCIEHNEALLEEAKMMNRLRHSRVVKLLGVIIEEGKYSLVMEYMEKGNLMHVLKAEMSTPLSVKGRIILEIIEGMCYLHGKGVIHKDLKPENILVDNDFHIKIADLGLASFKMWSKLNNEEHNELREVDGTAKKNGGTLYYMAPEHLNDVNAKPTEKSDVYSFAVVLWAIFANKEPYENAICEQQLIMCIKSGNRPDVDDITEYCPREIISLMKLCWEANPEARPTFPGIEEKFRPFYLSQLEESVEEDVKSLKKEYSNENAVVKRMQSLQLDCVAVPSSRSNSATEQPGSLHSSQGLGMGPVEESWFAPSLEHPQEENEPSLQSKLQDEANYHLYGSRMDRQTKQQPRQNVAYNREEERRRRVSHDPFAQQRPYENFQNTEGKGTAYSSAASHGNAVHQPSGLTSQPQVLYQNNGLYSSHGFGTRPLDPGTAGPRVWYRPIPSHMPSLHNIPVPETNYLGNTPTMPFSSLPPTDESIKYTIYNSTGIQIGAYNYMEIGGTSSSGSDGSGSGSGSLEIRAAFLEKENTALRTRAAELRKRVGRCRNIVSKYETRYGPL(huRIPK1(1-555).RR.DM; TH1023 without epitope tag) 102MQPDMSLDNIKMASSDLLEKTDLDSGGFGKVSLCYHRSHGFVILKKVYTGPNRAEYNEVLLEEGKMMHRLRHSRVVKLLGIIIEEGNYSLVMEYMEKGNLMHVLKTQIDVPLSLKGRIIVEAIEGMCYLHDKGVIHKDLKPENILVDRDFHIKIADLGVASFKTWSKLTKEKDNKQKEVSSTTKKNNGGTLYYMAPEHLNDINAKPTEKSDVYSFGIVLWAIFAKKEPYENVICTEQFVICIKSGNRPNVEEILEYCPREIISLMERCWQAIPEDRPTFLGIEEEFRPFYLSHFEEYVEEDVASLKKEYPDQSPVLQRMFSLQHDCVPLPPSRSNSEQPGSLHSSQGLQMGPVEESWFSSSPEYPQDENDRSVQAKLQEEASYHAFGIFAEKQTKPQPRQNEAYNREEERKRRVSHDPFAQQRARENIKSAGARGHSDPSTTSRGIAVQQLSWPATQTVWNNGLYNQHGFGTTGTGVWYPPNLSQMYSTYKTPVPETNIPGSTPTMPYFSGPVADDLIKYTIFNSSGIQIGNHNYMDVGLNSQPPNNTCKEESTSGGIKKEIEAIKKEQEAIKKKIEAIEKEIEA (msRIPK1(1-555).IZ.TM; TH1024without epitope tag) 103MQPDMSLDNIKMASSDLLEKTDLDSGGFGKVSLCYHRSHGFVILKKVYTGPNRAEYNEVLLEEGKMMHRLRHSRVVKLLGIIIEEGNYSLVMEYMEKGNLMHVLKTQIDVPLSLKGRIIVEAIEGMCYLHDKGVIHKDLKPENILVDRDFHIKIADLGVASFKTWSKLTKEKDNKQKEVSSTTKKNNGGTLYYMAPEHLNDINAKPTEKSDVYSFGIVLWAIFAKKEPYENVICTEQFVICIKSGNRPNVEEILEYCPREIISLMERCWQAIPEDRPTFLGIEEEFRPFYLSHFEEYVEEDVASLKKEYPDQSPVLQRMFSLQHDCVPLPPSRSNSEQPGSLHSSQGLQMGPVEESWFSSSPEYPQDENDRSVQAKLQEEASYHAFGIFAEKQTKPQPRQNEAYNREEERKRRVSHDPFAQQRARENIKSAGARGHSDPSTTSRGIAVQQLSWPATQTVWNNGLYNQHGFGTTGTGVWYPPNLSQMYSTYKTPVPETNIPGSTPTMPYFSGPVADDLIKYTIFNSSGIQIGNHNYMDVGLNSQPPNNTCKEESTSGSDGSGSGSGSITIRAAFLEKENTALRTEIAELEKEVGRCENIVSKYETRYGPL(msRIPK1(1-555).EE.DM; TH1025 without epitope tag) 104MQPDMSLDNIKMASSDLLEKTDLDSGGFGKVSLCYHRSHGFVILKKVYTGPNRAEYNEVLLEEGKMMHRLRHSRVVKLLGIIIEEGNYSLVMEYMEKGNLMHVLKTQIDVPLSLKGRIIVEAIEGMCYLHDKGVIHKDLKPENILVDRDFHIKIADLGVASFKTWSKLTKEKDNKQKEVSSTTKKNNGGTLYYMAPEHLNDINAKPTEKSDVYSFGIVLWAIFAKKEPYENVICTEQFVICIKSGNRPNVEEILEYCPREIISLMERCWQAIPEDRPTFLGIEEEFRPFYLSHFEEYVEEDVASLKKEYPDQSPVLQRMFSLQHDCVPLPPSRSNSEQPGSLHSSQGLQMGPVEESWFSSSPEYPQDENDRSVQAKLQEEASYHAFGIFAEKQTKPQPRQNEAYNREEERKRRVSHDPFAQQRARENIKSAGARGHSDPSTTSRGIAVQQLSWPATQTVWNNGLYNQHGFGTTGTGVWYPPNLSQMYSTYKTPVPETNIPGSTPTMPYFSGPVADDLIKYTIFNSSGIQIGNHNYMDVGLNSQPPNNTCKEESTSGSDGSGSGSGSLEIRAAFLEKENTALRTRAAELRKRVGRCRNIVSKYETRYGPL(msRIPK1(1-555).RR.DM; TH1026 without epitope tag) 105MSTASAASSSSSSSAGEMIEAPSQVLNFEEIDYKEIEVEEVVGRGAFGVVCKAKWRAKDVAIKQIESESERKAFIVELRQLSRVNHPNIVKLYGACLNPVCLVMEYAEGGSLYNVLHGAEPLPYYTAAHAMSWCLQCSQGVAYLHSMQPKALIHRDLKPPNLLLVAGGTVLKICDFGTACDIQTHMTNNKGSAAWMAPEVFEGSNYSEKCDVFSWGIILWEVITRRKPFDEIGGPAFRIMWAVHNGTRPPLIKNLPKPIESLMTRCWSKDPSQRPSMEEIVKIMTHLMRYFPGADEPLQYPCQEFGGGGGQSPTLTLQSTNTHTQSSSSSSDGGLFRSRPAHSLPPGEDGRVEPYVDFAEFYRLWSVDHGEQSVVTAP(human TAK1-TAB1; P4031 without epitope tag) 106MAALKSWLSRSVTSFFRYRQCLCVPVVANFKKRCFSELIRPWHKTVTIGFGVTLCAVPIAQKSEPHSLSSEALMRRAVSLVTDSTSTFLSQTTYALIEAITEYTKAVYTLTSLYRQYTSLLGKMNSEEEDEVWQVIIGARAEMTSKHQEYLKLETTWMTAVGLSEMAAEAAYQTGADQASITARNHIQLVKLQVEEVHQLSRKAETKLAEAQIEELRQKTQEEGEERAESEQEAYLRED (Diablo.1; without epitope tag) 107MAALKSWLSRSVTSFFRYRQCLCVPVVANFKKRCFSELIRPWHKTVTIGFGVTLCAVPIAQKSEPHSLSSEALMRRAVSLVTDSTSTFLSQTTYALIEAITEYTKAVYTLTSLYRQYTSLLGKMNLEEEDEVWQVIIGARAEMTSKHQEYLKLETTWMTAVGLSEMAAEAAYQTGADQASITARNHIQLVKLQVEEVHQLSRKAETKLAEAQIEELRQKTQEEGEERAESEQEAYLRED (Diablo.1(S126L); without epitope tag) 108MAVPIAQKSEPHSLSSEALMRRAVSLVTDSTSTFLSQTTYALIEAITEYTKAVYTLTSLYRQYTSLLGKMNSEEEDEVWQVIIGARAEMTSKHQEYLKLETTWMTAVGLSEMAAEAAYQTGADQASITARNHIQLVKLQVEEVHQLSRKAETKLAEAQIEELRQKTQEEGEERAESEQEAYLRED (Diablo.1(56-239); without epitope tag) 109MAVPIAQKSEPHSLSSEALMRRAVSLVTDSTSTFLSQTTYALIEAITEYTKAVYTLTSLYRQYTSLLGKMNLEEEDEVWQVIIGARAEMTSKHQEYLKLETTWMTAVGLSEMAAEAAYQTGADQASITARNHIQLVKLQVEEVHQLSRKAETKLAEAQIEELRQKTQEEGEERAESEQEAYLRED (Diablo.1(56-239/S126L); without epitopetag) 110MAALKSWLSRSVTSFFRYRQCLCVPVVANFKKRCFSELIRPWHKTVTIGFGVTLCAVPIAQAVYTLTSLYRQYTSLLGKMNSEEEDEVWQVIIGARAEMTSKHQEYLKLETTWMTAVGLSEMAAEAAYQTGADQASITARNHIQLVKLQVEEVHQLSRKAETKLAEAQIEELRQKTQEEGEERAESEQEAYLRED (Diablo.3; TH2003 withoutepitope tag) 111MAALKSWLSRSVTSFFRYRQCLCVPVVANFKKRCFSELIRPWHKTVTIGFGVTLCAVPIAQAVYTLTSLYRQYTSLLGKMNLEEEDEVWQVIIGARAEMTSKHQEYLKLETTWMTAVGLSEMAAEAAYQTGADQASITARNHIQLVKLQVEEVHQLSRKAETKLAEAQIEELRQKTQEEGEERAESEQEAYLRED (Diablo.3(S82L); TH2001without epitope tag) 112MAVPIAQAVYTLTSLYRQYTSLLGKMNSEEEDEVWQVIIGARAEMTSKHQEYLKLETTWMTAVGLSEMAAEAAYQTGADQASITARNHIQLVKLQVEEVHQLSRKAETKLAEAQIEELRQKTQEEGEERAESEQEAYLRED(Diablo.3(56-195); TH2002 without epitope tag) 113MAVPIAQAVYTLTSLYRQYTSLLGKMNLEEEDEVWQVIIGARAEMTSKHQEYLKLETTWMTAVGLSEMAAEAAYQTGADQASITARNHIQLVKLQVEEVHQLSRKAETKLAEAQIEELRQKTQEEGEERAESEQEAYLRED(Diablo.3(56-195/S82L); without epitope tag) 114MAAVILESIFLKRSQQKKKTSPLNFKKRLFLLTVHKLSYYKYDFERGRRGSKKGSIDVEKITCVETVVPEKNPPPERQIPRRGEESSEMEQISIIERFPYPFQVVYDEGPLYVFSPTEELRKRWIHQLKNVIRYNSDLVQKYHPCFWIDGQYLCCSQTAKNAMGCQILENRNGSLKPGSSHRKTKKPLPPTPEEDQILKKPLPPEPAAAPVSTSELKKVVALYDYMPMNANDLQLRKGDEYFILEESNLPWWRARDKNGQEGYIPSNYVTEAEDSIEMYEWYSKHMTRSQAEQLLKQEGKEGGFIVRDSSKAGKYTVSVFAKSTGDPQGVIRHYVVCSTPOSQYYLAEKHLFSTIPELINYHQHNSAGLISRLKYPVSQQNKNAPSTAGLGYGSWEIDPKDLTFLKELGTGQFGVVKYGKWRGQYDVAIKMIKEGSMSEDEFIEEAKVMMNLSHEKLVQLYGVCTKQRPIFIITEYMANGCLLNYLREMRHRFQTQQLLEMCKDVCEAMEYLESKQFLHRDLAARNCLVNDQGVVKVSDFGLSRYVLDDEYTSSVGSKFPVRWSPPEVLMYSKFSSKSDIWAFGVLMWEIYSLGKMPYERFTNSETAEHIAQGLRLYRPHLASEKVYTIMYSCWHEKADERPTFKILLSNILDVMDEES (Btk(E41K); P4029 without epitopetag) 115MVTHSKFPAAGMSRPLDTSLRLKTFSSKSEYQLVVNAVRKLQESGFYWSAVTGGEANLLLSAEPAGTFLIRDSSDQRHFFTLSVKTQSGTKNLRIQCEGGSFSLQSDPRSTQPVPRFDCVLKLVHHYMPPPGAPSFRSPPTEPSSEVPEQPSAQPLPGSPPRRAYYIYSGGEKIPLVLSRPLSSNVATLQHLCRKTVNGHLDSYEKVTQLPGPIREFLDQYDAPL(SOCS3; P4030 without epitope tag) 116MRMKQIEDKIEEILSKIYHINEIARIKKLIGEADQTSGNYLNMQDSQGVLSSFPAPQAVQDNPAMPTSSGSEGNVKLCSLEEAQRIWKQKSAEIYPIMDKSSRTRLALIICNEEFDSIPRRTGAEVDITGMTMLLQNLGYSVDVKKNLTASDMTTELEAFAHRPEHKTSDSTFLVFMSHGIREGICGKKHSEQVPDILQLNAIFNMLNTKNCPSLKDKPKVIIIQACRGDSPGVVWFKDSVGVSGNLSLPTTEEFEDDAIKKAHIEKDFIAFCSSTPDNVSWRHPTMGSVFIGRLIEHMQEYACSCDVEEIFRKVRFSFEQPDGRAQMPTTERVTLTRCFYLFPGH (IZ_hsCASP1 (self-activating humanCaspase 1); P2024 without epitope tag) 117MRMKQLEDKIEELLSKIYHLENEIARLKKLIGEADQTSGNYLNMQDSQGVLSSFPAPQAVQDNPAMPTSSGSEGNVKLCSLEEAQRIWKQKSAEIYPIMDKSSRTRLALIICNEEFDSIPRRTGAEVDITGMTMLLQNLGYSVDVKIKNLTASDMTTELEAFAHRPEHKTSDSTFLVFMSHGIREGICGKKHSEQVPDILQLNAIFNMLNTKNCPSLKDKPKVIIIQACRGDSPGVVWFKDSVGVSGNLSLPTTEEFEDDAIKKAHIEKDFIAFCSSTPDNVSWRHPTMGSVFIGRLIEHMQEYACSCDVEEIFRKVRFSFEQPDGRAQMPTTERVTLTRCFYLFPGH (DM_hsCASP1 (self-activating humanCaspase 1); P2025 without epitope tag) 118MRMKQIEDKIEEILSKIYHIENEIARIKKLIGERSAPSAETFVATEDSKGGHPSSSETKEEQNKEDGTFPGLTGTLKFCPLEKAQKLWKENPSEIYPIMNTTTRTRLALIICNTEFQHLSPRVGAQVDLREMKLLLEDLGYTVKVKENLTALEMVKEVKEFAACPEHKTSDSTFLVFMSHGIQEGICGTTYSNEVSDILKVDTIFQMMNTLKCPSLKDKPKVIIIQACRGEKQGVVLLKDSVRDSEEDFLTDAIFEDDGIKKAHIEKDFIAFCSSTPDNVSWRHPVRGSLFIESLIKHMKEYAWSCDLEDIFRKVRFSFEQPEFRLQMPTADRVTLTKRFYLFPGH (IZ_mmCASP1 (self-activating mouse Caspase 1);P2026 without epitope tag) 119MRMKQLEDKIEELLSKIYHLENEIARLKKLIGERSARSAETFVATEDSKGGHPSSSETKEEQNKEDGTFPGLTGTLKFCPLEKAQKLWKENPSEIYPIMNTTTRTRLALIICNTEFQHLSPRVGAQVDLREMKLLLEDLGYTVKVKENLTALEMVKEVKEFAACPEHKTSDSTFLVFMSHGIQEGICGTTYSNEVSDILKVDTIFQMMNTLKCPSLKDKPKVIIIQACRGEKQGVVLLKDSVRDSEEDFLTDAIFEDDGIKKAHIEKDFIAFCSSTPDNVSWRHPVRGSLFIESLIKHMKEYAWSCDLEDIFRKVRFSFEQPEFRLQMPTADRVTLTKRFYLFPGH (DM_mmCASP1 (self-activating mouse Caspase 1);P2027 without epitope tag) 120MHHHHHHHHHHGKPIPNPLLGLDSTGIPVHLELASMTNMELMSSIVHQQVFPTEAGQSLVISASIIVFNLLELEGDYRGRVLELFRAAQLANDVVLQIMELCGATR (ADR concatemer with HIS tag) 121 VVGADGVGK(KRAS G12D 9mer) 122 VVGAVGVGK (KRAS G12V 9mer) 123 VGAGDVGKS (KRAS G13D9mer) 124 VVGACGVGK (KRAS G12C 9mer) 125 MKLVVVGACGVGKSA (KRAS G12C15mer) 126ATGACCGAGTACAAGCTGGTGGTGGTGGGCGCCGACGGCGTGGGCAAGAGCGCCCTGACCATCCAGCTGATCCAG (KRAS G12D 25mer nucleotide sequence) 127ATGACCGAGTACAAGCTGGTGGTGGTGGGCGCCGTGGGCGTGGGCAAGAGCGCCCTGACCATCCAGCTGATCCAG (KRAS G12V 25mer nucleotide sequence) 128ATGACCGAGTACAAGCTGGTGGTGGTGGGCGCCGGCGACGTGGGCAAGAGCGCCCTGACCATCCAGCTGATCCAG (KRAS G13D 25mer nucleotide sequence) 129ATGACCGAGTACAAGTTAGTGGTTGTGGGCGCCGACGGCGTGGGCAAGAGCGCCCTCACCATCCAGCTTATCCAGATGACGGAATATAAGTTAGTAGTAGTGGGAGCCGACGGTGTCGGCAAGTCCGCTTTGACCATTCAACTTATTCAGATGACAGAGTATAAGCTGGTCGTTGTAGGCGCAGACGGCGTTGGAAAGTCGGCACTGACGATCCAGTTGATCCAG (KRAS G12D 25mer^3 nucleotide sequence) 130ATGACCGAGTACAAGCTCGTCGTGGTGGGCGCCGTGGGCGTGGGCAAGAGCGCCCTAACCATCCAGTTGATCCAGATGACCGAATATAAGCTCGTGGTAGTCGGAGCGGTGGGCGTTGGCAAGTCAGCGCTAACAATACAACTAATCCAAATGACCGAATACAAGCTAGTTGTAGTCGGTGCCGTCGGCGTTGGAAAGTCAGCCCTTACAATTCAGCTCATTCAG (KRAS G12V 25mer^3 nucleotide sequence) 131ATGACCGAGTACAAGCTCGTAGTGGTTGGCGCCGGCGACGTGGGCAAGAGCGCCCTAACCATCCAGCTCATCCAGATGACAGAATATAAGCTTGTGGTTGTGGGAGCAGGAGACGTGGGAAAGAGTGCGTTGACGATTCAACTCATACAGATGACCGAATACAAGTTGGTGGTGGTCGGCGCAGGTGACGTTGGTAAGTCTGCACTAACTATACAACTGATCCAG (KRAS G13D 25mer^3 nucleotide sequence) 132ATGACCGAGTACAAGCTGGTGGTGGTGGGCGCCTGCGGCGTGGGCAAGAGCGCCCTGACCATCCAGCTGATCCAG (KRAS G12C 25mer nucleotide sequence) 133ATGACCGAGTACAAGCTGGTGGTGGTGGGCGCCGGCGGCGTGGGCAAGAGCGCCCTGACCATCCAGCTGATCCAG (KRAS WT 25mer nucleotide sequence) 134GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACC (5′ UTR sequence; nopromoter) 135MTEYKLVVVGADGVGKSALTIQLIQMTEYKLVVVGAVGVGKSALTIQLIQMTEYKLVVVGAGDVGKSALTIQLIQ(KRAS(G12D G12V G13D) 75mer “3MUT” aa. seq) 136ATGACCGAGTACAAGCTCGTTGTAGTCGGCGCCGACGGCGTGGGCAAGAGCGCCTTGACCATCCAGTTGATCCAGATGACCGAATATAAGTTGGTGGTGGTAGGCGCAGTGGGAGTTGGCAAGTCAGCACTCACAATTCAGCTCATTCAAATGACAGAATACAAGTTAGTCGTTGTAGGAGCAGGCGACGTCGGCAAGAGTGCCTTAACCATTCAACTAATCCAG (KRAS(G12D G12V G13D) 75mer “3MUT” nt. seq) 137MTEYKLVVVGADGVGKSALTIQLIQMTEYKLVVVGAVGVGKSALTIQLIQMTEYKLVVVGAGDVGKSALTIQLIQMTEYKLVVVGACGVGKSALTIQLIQ (KRAS(G12D G12V G13D G12C) 100mer “4MUT” aa. seq)138ATGACCGAGTACAAGCTCGTGGTCGTCGGCGCCGACGGGGTAGGCAAGTCCGCTCTGACCATTCAGCTCATCCAGATGACGGAGTACAAACTCGTGGTAGTGGGAGCCGTGGGTGTGGGCAAGAGCGCGCTCACCATCCAACTCATCCAAATGACCGAATATAAACTCGTCGTGGTGGGAGCCGGCGACGTGGGAAAGAGCGCCCTTACCATCCAGTTAATCCAGATGACAGAATACAAGCTGGTGGTGGTCGGTGCCTGCGGCGTGGGTAAGTCCGCCCTGACAATCCAGCTGATCCAG (KRAS(G12D G12V G13D G12C) 100mer “4MUT” nt. seq) 139ATGCCCCACAGTAGCCTCCACCCCAGCATCCCCTGCCCCAGAGGCCACGGCGCACAGAAGGCCGCCCTGGTGCTGCTGAGCGCCTGTCTGGTGACCCTGTGGGGTCTGGGCGAGCCCCCCGAGCACACCCTGCGGTACCTCGTGCTGCATCTGGCCAGCCTGCAGCTGGGCCTGCTGCTGAACGGCGTGTGCAGCCTGGCCGAAGAGCTGAGACACATCCACAGCAGATACAGAGGCTCCTACTGGAGAACCGTCAGAGCCTGCCTCGGCTGTCCCCTGAGAAGAGGCGCCCTGCTGCTCCTGAGCATCTACTTCTACTACAGCCTGCCCAACGCCGTGGGCCCCCCCTTCACCTGGATGCTGGCCCTGCTGGGCCTGAGCCAGGCCCTGAACATCCTGCTGGGCCTGAAGGGCTTGGCCCCCGCCGAGATCTCCGCCGTGTGCGAGAAGGGCAACTTCAACATGGCCCATGGCCTTGCCTGGTCCTACTACATCGGCTACCTGAGACTGATCCTGCCCGAGCTGCAGGCCAGAATCAGAACCTACAACCAGCACTACAACAACCTGCTGAGAGGCGCCGTGAGCCAAAGACTGTACATCCTGCTGCCCCTGGACTGCGGCGTGCCCGACAACCTTAGCATGGCCGACCCCAACATCAGATTCCTGGACAAGCTGCCCCAGCAGACCGGCGACCACGCCGGCATCAAGGACAGAGTGTACAGCAACAGCATCTACGAGCTGCTGGAGAACGGCCAGAGAGCCGGCACCTGCGTGCTGGAGTACGCCACCCCCCTGCAGACCCTGTTCGCCATGAGCCAGTACAGCCAGGCCGGCTTCAGCAGAGAGGACAGACTGGAGCAAGCCAAGCTGTTCTGCAGAACCCTGGAGGACATCCTGGCGGACGCCCCCGAGAGCCAAAACAACTGCAGACTGATCGCCTACCAGGAGCCCGCCGACGACAGCAGCTTCAGCCTGAGCCAGGAAGTGCTGAGACACCTGAGACAGGAAGAGAAGGAGGAGGTGACCGTGGGAAGCCTGAAGACCAGCGCCGTGCCCAGCACCAGCACCATGAGCCAGGAGCCCGAGCTGCTGATCAGCGGCATGGAGAAGCCCCTGCCCCTGAGAACCGACTTCAGC (huSTING(V155M); no epitope tag;nucleotide sequence) 140ATGCCTCACAGCAGCCTGCACCCTAGCATCCCTTGCCCTAGAGGCCACGGCGCCCAGAAGGCCGCCCTGGTGCTGCTGAGCGCCTGCCTGGTGACCCTGTGGGGCCTGGGCGAGCCTCCTGAGCACACCCTGAGATACCTGGTGCTGCACCTGGCCAGCCTGCAGCTGGGCCTGCTGCTGAACGGCGTGTGCAGCCTGGCCGAGGAGCTGAGACACATCCACAGCAGATACAGAGGCAGCTACTGGAGAACCGTGAGAGCCTGCCTGGGCTGCCCTCTGAGAAGAGGCGCCCTGCTGCTGCTGAGCATCTACTTCTACTACAGCCTGCCTAACGCCGTGGGCCCTCCTTTCACCTGGATGCTGGCCCTGCTGGGCCTGAGCCAGGCCCTGAACATCCTGCTGGGCCTGAAGGGCCTGGCCCCTGCCGAGATCAGCGCCGTGTGCGAGAAGGGCAACTTCAACGTGGCCCACGGCCTGGCCTGGAGCTACTACATCGGCTACCTGAGACTGATCCTGCCTGAGCTGCAGGCCAGAATCAGAACCTACAACCAGCACTACAACAACCTGCTGAGAGGCGCCGTGAGCCAGAGACTGTACATCCTGCTGCCTCTGGACTGCGGCGTGCCTGACAACCTGAGCATGGCCGACCCTAACATCAGATTCCTGGACAAGCTGCCTCAGCAGACCGGCGACCACGCCGGCATCAAGGACAGAGTGTACAGCAACAGCATCTACGAGCTGCTGGAGAACGGCCAGAGAGCCGGCACCTGCGTGCTGGAGTACGCCACCCCTCTGCAGACCCTGTTCGCCATGAGCCAGTACAGCCAGGCCGGCTTCAGCAGAGAGGACACCCTGGAGCAGGCCAAGCTGTTCTGCAGAACCCTGGAGGACATCCTGGCCGACGCCCCTGAGAGCCAGAACAACTGCAGACTGATCGCCTACCAGGAGCCTGCCGACGACAGCAGCTTCAGCCTGAGCCAGGAGGTGCTGAGACACCTGAGACAGGAGGAGAAGGAGGAGGTGACCGTGGGCAGCCTGAAGACCAGCGCCGTGCCTAGCACCAGCACCATGAGCCAGGAGCCTGAGCTGCTGATCAGCGGCATGGAGAAGCCTCTGCCTCTGAGAACCGACTTCAGC (Hu STING(R284T); no epitope tag;nucleotide sequence) 141ATGCCCCACAGCAGCCTGCACCCCTCCATCCCCTGTCCCAGAGGCCACGGCGCCCAGAAGGCCGCCCTGGTGCTGCTGAGCGCCTGCCTGGTGACCTTATGGGGCCTGGGCGAGCCCCCCGAGCACACCCTGAGATACCTGGTCCTGCACCTGGCCAGCCTCCAGCTGGGCCTGCTGCTCAACGGCGTGTGTAGCCTGGCCGAGGAGCTGAGACACATCCACAGCAGATACAGAGGCAGCTACTGGAGAACCGTGAGAGCCTGCCTGGGTTGCCCACTGAGAAGAGGAGCTCTGCTGCTGCTGAGCATCTACTTCTACTACTCGCTGCCCAACGCTGTGGGCCCCCCCTTCACCTGGATGCTGGCCCTGCTGGGTCTGAGCCAGGCCCTGAACATCCTCCTGGGCCTGAAGGGCCTGGCCCCCGCCGAGATAAGCGCCGTTTGCGAGAAGGGCAACTTCAACGTGGCCCATGGCCTGGCCTGGAGCTACTACATCGGCTACTTACGCCTGATCCTGCCCGAGCTGCAGGCCAGAATCAGAACCTACAACCAGCATTACAACAACCTGCTGAGAGGCGCCGTGAGCCAGAGACTGTATATCCTGCTGCCCCTGGACTGCGGCGTGCCCGACAACCTGAGCATGGCCGACCCCAACATCAGATTCCTGGACAAGCTCCCCCAGCAGACCGGCGACCACGCCGGAATCAAAGACAGAGTGTATAGCAACAGCATCTACGAGCTGCTGGAGAACGGCCAGAGAGCCGGCACCTGCGTACTGGAGTACGCCACCCCCTTGCAGACCCTGTTTGCCATGAGCCAGTACAGCCAGGCCGGCTTCAGCAGAGAGGACATGCTGGAGCAGGCCAAGCTGTTCTGCAGAACCCTGGAGGACATCCTGGCCGACGCCCCCGAGAGCCAGAACAACTGCAGACTGATCGCCTACCAAGAGCCCGCCGACGACAGCAGCTTCAGCTTAAGCCAGGAGGTGCTGAGACATCTGAGACAGGAGGAGAAGGAGGAGGTGACCGTGGGCAGCCTCAAGACCAGCGCTGTGCCCTCTACCAGCACCATGAGCCAGGAGCCCGAGCTGCTGATCAGCGGCATGGAGAAGCCCCTGCCCCTGAGAACAGACTTCAGC (hu STING (R284M); no epitope tag;nucleotide sequence) 142ATGCCCCATAGCAGCCTGCACCCCAGCATCCCCTGCCCCAGAGGCCACGGCGCCCAGAAGGCCGCCCTGGTCCTGCTGAGCGCATGCCTGGTCACCCTGTGGGGCCTGGGCGAGCCCCCCGAGCACACCCTGAGATACCTGGTGCTGCACCTCGCCAGCCTGCAGCTGGGCCTGCTGCTGAACGGCGTGTGCAGCCTGGCCGAGGAGCTGAGACACATCCACAGCAGATATAGAGGCAGCTACTGGAGAACCGTGAGAGCTTGCCTCGGCTGCCCCCTGAGAAGAGGCGCCCTGCTGCTGCTGAGCATCTACTTTTACTACAGCCTGCCCAACGCTGTGGGCCCCCCTTTCACGTGGATGCTCGCCCTGCTGGGACTGAGCCAGGCCCTGAACATCCTGCTGGGCCTTAAGGGCCTAGCCCCCGCCGAGATCAGCGCCGTGTGCGAGAAGGGCAACTTCAATGTGGCCCACGGCCTGGCCTGGAGCTACTACATCGGCTACCTGAGACTGATCCTGCCCGAGCTGCAGGCCAGAATCAGAACCTACAATCAGCACTACAACAACCTGCTGAGAGGCGCCGTGAGCCAGAGACTGTACATCCTGCTGCCCCTGGACTGCGGCGTGCCCGACAACCTCAGCATGGCCGACCCCAACATCAGATTCCTGGACAAGCTGCCCCAGCAGACCGGCGACCACGCCGGCATCAAGGATCGCGTGTACAGCAACAGCATCTACGAGCTGCTGGAAAACGGCCAGAGAGCCGGAACCTGCGTGCTGGAGTACGCCACACCCCTGCAGACCCTGTTCGCCATGAGCCAGTACAGCCAGGCCGGCTTCAGCAGAGAGGACAAGCTGGAGCAGGCCAAGCTGTTCTGCAGAACCCTGGAGGATATCCTCGCCGACGCCCCCGAGAGCCAGAACAACTGCAGGCTGATCGCGTACCAGGAGCCCGCTGACGACAGCAGCTTTAGCCTGAGCCAGGAGGTGCTGAGACATCTGCGTCAAGAGGAAAAGGAGGAGGTGACCGTGGGCTCCCTGAAGACCAGCGCCGTGCCCAGCACCAGCACCATGAGCCAGGAGCCCGAGCTGCTGATCAGCGGCATGGAGAAGCCACTGCCCCTCAGAACCGACTTCAGC (Hu STING (R284K); no epitope tag;nucleotide sequence) 143ATGCCTCACAGCAGCCTGCACCCTAGCATCCCTTGCCCTAGAGGCCACGGCGCCCAGAAGGCCGCCCTGGTGCTGCTGAGCGCCTGCCTGGTGACCCTGTGGGGCCTGGGCGAGCCTCCTGAGCACACCCTGAGATACCTGGTGCTGCACCTGGCCAGCCTGCAGCTGGGCCTGCTGCTGAACGGCGTGTGCAGCCTGGCCGAGGAGCTGAGACACATCCACAGCAGATACAGAGGCAGCTACTGGAGAACCGTGAGAGCCTGCCTGGGCTGCCCTCTGAGAAGAGGCGCCCTGCTGCTGCTGAGCATCTACTTCTACTACAGCCTGCCTAACGCCGTGGGCCCTCCTTTCACCTGGATGCTGGCCCTGCTGGGCCTGAGCCAGGCCCTGAACATCCTGCTGGGCCTGAAGGGCCTGGCCCCTGCCGAGATCAGCGCCGTGTGCGAGAAGGGCAACTTCAGCGTGGCCCACGGCCTGGCCTGGAGCTACTACATCGGCTACCTGAGACTGATCCTGCCTGAGCTGCAGGCCAGAATCAGAACCTACAACCAGCACTACAACAACCTGCTGAGAGGCGCCGTGAGCCAGAGACTGTACATCCTGCTGCCTCTGGACTGCGGCGTGCCTGACAACCTGAGCATGGCCGACCCTAACATCAGATTCCTGGACAAGCTGCCTCAGCAGACCGGCGACCACGCCGGCATCAAGGACAGAGTGTACAGCAACAGCATCTACGAGCTGCTGGAGAACGGCCAGAGAGCCGGCACCTGCGTGCTGGAGTACGCCACCCCTCTGCAGACCCTGTTCGCCATGAGCCAGTACAGCCAGGCCGGCTTCAGCAGAGAGGACAGACTGGAGCAGGCCAAGCTGTTCTGCAGAACCCTGGAGGACATCCTGGCCGACGCCCCTGAGAGCCAGAACAACTGCAGACTGATCGCCTACCAGGAGCCTGCCGACGACAGCAGCTTCAGCCTGAGCCAGGAGGTGCTGAGACACCTGAGACAGGAGGAGAAGGAGGAGGTGACCGTGGGCAGCCTGAAGACCAGCGCCGTGCCTAGCACCAGCACCATGAGCCAGGAGCCTGAGCTGCTGATCAGCGGCATGGAGAAGCCTCTGCCTCTGAGAACCGACTTCAGC (Hu STING(N154S); no epitope tag;nucleotide sequence) 144ATGCCTCACAGCAGCCTGCACCCTAGCATCCCTTGCCCTAGAGGCCACGGCGCCCAGAAGGCCGCCCTGGTGCTGCTGAGCGCCTGCCTGGTGACCCTGTGGGGCCTGGGCGAGCCTCCTGAGCACACCCTGAGATACCTGGTGCTGCACCTGGCCAGCCTGCAGCTGGGCCTGCTGCTGAACGGCGTGTGCAGCCTGGCCGAGGAGCTGAGACACATCCACAGCAGATACAGAGGCAGCTACTGGAGAACCGTGAGAGCCTGCCTGGGCTGCCCTCTGAGAAGAGGCGCCCTGCTGCTGCTGAGCATCTACTTCTACTACAGCCTGCCTAACGCCGTGGGCCCTCCTTTCACCTGGATGCTGGCCCTGCTGGGCCTGAGCCAGGCCCTGAACATCCTGCTGGGCCTGAAGGGCCTGGCCCCTGCCGAGATCAGCGCCCTGTGCGAGAAGGGCAACTTCAACGTGGCCCACGGCCTGGCCTGGAGCTACTACATCGGCTACCTGAGACTGATCCTGCCTGAGCTGCAGGCCAGAATCAGAACCTACAACCAGCACTACAACAACCTGCTGAGAGGCGCCGTGAGCCAGAGACTGTACATCCTGCTGCCTCTGGACTGCGGCGTGCCTGACAACCTGAGCATGGCCGACCCTAACATCAGATTCCTGGACAAGCTGCCTCAGCAGACCGGCGACCACGCCGGCATCAAGGACAGAGTGTACAGCAACAGCATCTACGAGCTGCTGGAGAACGGCCAGAGAGCCGGCACCTGCGTGCTGGAGTACGCCACCCCTCTGCAGACCCTGTTCGCCATGAGCCAGTACAGCCAGGCCGGCTTCAGCAGAGAGGACAGACTGGAGCAGGCCAAGCTGTTCTGCAGAACCCTGGAGGACATCCTGGCCGACGCCCCTGAGAGCCAGAACAACTGCAGACTGATCGCCTACCAGGAGCCTGCCGACGACAGCAGCTTCAGCCTGAGCCAGGAGGTGCTGAGACACCTGAGACAGGAGGAGAAGGAGGAGGTGACCGTGGGCAGCCTGAAGACCAGCGCCGTGCCTAGCACCAGCACCATGAGCCAGGAGCCTGAGCTGCTGATCAGCGGCATGGAGAAGCCTCTGCCTCTGAGAACCGACTTCAGC (Hu STING(V147L); no epitope tag;nucleotide sequence) 145ATGCCTCACAGCAGCCTGCACCCTAGCATCCCTTGCCCTAGAGGCCACGGCGCCCAGAAGGCCGCCCTGGTGCTGCTGAGCGCCTGCCTGGTGACCCTGTGGGGCCTGGGCGAGCCTCCTGAGCACACCCTGAGATACCTGGTGCTGCACCTGGCCAGCCTGCAGCTGGGCCTGCTGCTGAACGGCGTGTGCAGCCTGGCCGAGGAGCTGAGACACATCCACAGCAGATACAGAGGCAGCTACTGGAGAACCGTGAGAGCCTGCCTGGGCTGCCCTCTGAGAAGAGGCGCCCTGCTGCTGCTGAGCATCTACTTCTACTACAGCCTGCCTAACGCCGTGGGCCCTCCTTTCACCTGGATGCTGGCCCTGCTGGGCCTGAGCCAGGCCCTGAACATCCTGCTGGGCCTGAAGGGCCTGGCCCCTGCCGAGATCAGCGCCGTGTGCGAGAAGGGCAACTTCAACGTGGCCCACGGCCTGGCCTGGAGCTACTACATCGGCTACCTGAGACTGATCCTGCCTGAGCTGCAGGCCAGAATCAGAACCTACAACCAGCACTACAACAACCTGCTGAGAGGCGCCGTGAGCCAGAGACTGTACATCCTGCTGCCTCTGGACTGCGGCGTGCCTGACAACCTGAGCATGGCCGACCCTAACATCAGATTCCTGGACAAGCTGCCTCAGCAGACCGGCGACCACGCCGGCATCAAGGACAGAGTGTACAGCAACAGCATCTACGAGCTGCTGGAGAACGGCCAGAGAGCCGGCACCTGCGTGCTGGAGTACGCCACCCCTCTGCAGACCCTGTTCGCCATGAGCCAGTACAGCCAGGCCGGCTTCAGCAGAGAGGACAGACTGGAGCAGGCCAAGCTGTTCTGCAGAACCCTGGAGGACATCCTGGCCGACGCCCCTGAGAGCCAGAACAACTGCAGACTGATCGCCTACCAGCAGCCTGCCGACGACAGCAGCTTCAGCCTGAGCCAGGAGGTGCTGAGACACCTGAGACAGGAGGAGAAGGAGGAGGTGACCGTGGGCAGCCTGAAGACCAGCGCCGTGCCTAGCACCAGCACCATGAGCCAGGAGCCTGAGCTGCTGATCAGCGGCATGGAGAAGCCTCTGCCTCTGAGAACCGACTTCAGC (Hu STING (E315Q); no epitope tag;nucleotide sequence) 146ATGCCTCACAGCAGCCTGCACCCTAGCATCCCTTGCCCTAGAGGCCACGGCGCCCAGAAGGCCGCCCTGGTGCTGCTGAGCGCCTGCCTGGTGACCCTGTGGGGCCTGGGCGAGCCTCCTGAGCACACCCTGAGATACCTGGTGCTGCACCTGGCCAGCCTGCAGCTGGGCCTGCTGCTGAACGGCGTGTGCAGCCTGGCCGAGGAGCTGAGACACATCCACAGCAGATACAGAGGCAGCTACTGGAGAACCGTGAGAGCCTGCCTGGGCTGCCCTCTGAGAAGAGGCGCCCTGCTGCTGCTGAGCATCTACTTCTACTACAGCCTGCCTAACGCCGTGGGCCCTCCTTTCACCTGGATGCTGGCCCTGCTGGGCCTGAGCCAGGCCCTGAACATCCTGCTGGGCCTGAAGGGCCTGGCCCCTGCCGAGATCAGCGCCGTGTGCGAGAAGGGCAACTTCAACGTGGCCCACGGCCTGGCCTGGAGCTACTACATCGGCTACCTGAGACTGATCCTGCCTGAGCTGCAGGCCAGAATCAGAACCTACAACCAGCACTACAACAACCTGCTGAGAGGCGCCGTGAGCCAGAGACTGTACATCCTGCTGCCTCTGGACTGCGGCGTGCCTGACAACCTGAGCATGGCCGACCCTAACATCAGATTCCTGGACAAGCTGCCTCAGCAGACCGGCGACCACGCCGGCATCAAGGACAGAGTGTACAGCAACAGCATCTACGAGCTGCTGGAGAACGGCCAGAGAGCCGGCACCTGCGTGCTGGAGTACGCCACCCCTCTGCAGACCCTGTTCGCCATGAGCCAGTACAGCCAGGCCGGCTTCAGCAGAGAGGACAGACTGGAGCAGGCCAAGCTGTTCTGCAGAACCCTGGAGGACATCCTGGCCGACGCCCCTGAGAGCCAGAACAACTGCAGACTGATCGCCTACCAGGAGCCTGCCGACGACAGCAGCTTCAGCCTGAGCCAGGAGGTGCTGAGACACCTGAGACAGGAGGAGAAGGAGGAGGTGACCGTGGGCAGCCTGAAGACCAGCGCCGTGCCTAGCACCAGCACCATGAGCCAGGAGCCTGAGCTGCTGATCAGCGGCATGGAGAAGCCTCTGCCTCTGGCCACCGACTTCAGC (Hu STING (R375A); no epitope tag;nucleotide sequence) 147ATGCCTCACAGCAGCCTGCACCCTAGCATCCCTTGCCCTAGAGGCCACGGCGCCCAGAAGGCCGCCCTGGTGCTGCTGAGCGCCTGCCTGGTGACCCTGTGGGGCCTGGGCGAGCCTCCTGAGCACACCCTGAGATACCTGGTGCTGCACCTGGCCAGCCTGCAGCTGGGCCTGCTGCTGAACGGCGTGTGCAGCCTGGCCGAGGAGCTGAGACACATCCACAGCAGATACAGAGGCAGCTACTGGAGAACCGTGAGAGCCTGCCTGGGCTGCCCTCTGAGAAGAGGCGCCCTGCTGCTGCTGAGCATCTACTTCTACTACAGCCTGCCTAACGCCGTGGGCCCTCCTTTCACCTGGATGCTGGCCCTGCTGGGCCTGAGCCAGGCCCTGAACATCCTGCTGGGCCTGAAGGGCCTGGCCCCTGCCGAGATCAGCGCCCTGTGCGAGAAGGGCAACTTCAGCATGGCCCACGGCCTGGCCTGGAGCTACTACATCGGCTACCTGAGACTGATCCTGCCTGAGCTGCAGGCCAGAATCAGAACCTACAACCAGCACTACAACAACCTGCTGAGAGGCGCCGTGAGCCAGAGACTGTACATCCTGCTGCCTCTGGACTGCGGCGTGCCTGACAACCTGAGCATGGCCGACCCTAACATCAGATTCCTGGACAAGCTGCCTCAGCAGACCGGCGACCACGCCGGCATCAAGGACAGAGTGTACAGCAACAGCATCTACGAGCTGCTGGAGAACGGCCAGAGAGCCGGCACCTGCGTGCTGGAGTACGCCACCCCTCTGCAGACCCTGTTCGCCATGAGCCAGTACAGCCAGGCCGGCTTCAGCAGAGAGGACAGACTGGAGCAGGCCAAGCTGTTCTGCAGAACCCTGGAGGACATCCTGGCCGACGCCCCTGAGAGCCAGAACAACTGCAGACTGATCGCCTACCAGGAGCCTGCCGACGACAGCAGCTTCAGCCTGAGCCAGGAGGTGCTGAGACACCTGAGACAGGAGGAGAAGGAGGAGGTGACCGTGGGCAGCCTGAAGACCAGCGCCGTGCCTAGCACCAGCACCATGAGCCAGGAGCCTGAGCTGCTGATCAGCGGCATGGAGAAGCCTCTGCCTCTGAGAACCGACTTCAGC (Hu STING(V147L/N154S/V155M); noepitope tag; nucleotide sequence) 148ATGCCTCACAGCAGCCTGCACCCTAGCATCCCTTGCCCTAGAGGCCACGGCGCCCAGAAGGCCGCCCTGGTGCTGCTGAGCGCCTGCCTGGTGACCCTGTGGGGCCTGGGCGAGCCTCCTGAGCACACCCTGAGATACCTGGTGCTGCACCTGGCCAGCCTGCAGCTGGGCCTGCTGCTGAACGGCGTGTGCAGCCTGGCCGAGGAGCTGAGACACATCCACAGCAGATACAGAGGCAGCTACTGGAGAACCGTGAGAGCCTGCCTGGGCTGCCCTCTGAGAAGAGGCGCCCTGCTGCTGCTGAGCATCTACTTCTACTACAGCCTGCCTAACGCCGTGGGCCCTCCTTTCACCTGGATGCTGGCCCTGCTGGGCCTGAGCCAGGCCCTGAACATCCTGCTGGGCCTGAAGGGCCTGGCCCCTGCCGAGATCAGCGCCCTGTGCGAGAAGGGCAACTTCAGCATGGCCCACGGCCTGGCCTGGAGCTACTACATCGGCTACCTGAGACTGATCCTGCCTGAGCTGCAGGCCAGAATCAGAACCTACAACCAGCACTACAACAACCTGCTGAGAGGCGCCGTGAGCCAGAGACTGTACATCCTGCTGCCTCTGGACTGCGGCGTGCCTGACAACCTGAGCATGGCCGACCCTAACATCAGATTCCTGGACAAGCTGCCTCAGCAGACCGGCGACCACGCCGGCATCAAGGACAGAGTGTACAGCAACAGCATCTACGAGCTGCTGGAGAACGGCCAGAGAGCCGGCACCTGCGTGCTGGAGTACGCCACCCCTCTGCAGACCCTGTTCGCCATGAGCCAGTACAGCCAGGCCGGCTTCAGCAGAGAGGACATGCTGGAGCAGGCCAAGCTGTTCTGCAGAACCCTGGAGGACATCCTGGCCGACGCCCCTGAGAGCCAGAACAACTGCAGACTGATCGCCTACCAGGAGCCTGCCGACGACAGCAGCTTCAGCCTGAGCCAGGAGGTGCTGAGACACCTGAGACAGGAGGAGAAGGAGGAGGTGACCGTGGGCAGCCTGAAGACCAGCGCCGTGCCTAGCACCAGCACCATGAGCCAGGAGCCTGAGCTGCTGATCAGCGGCATGGAGAAGCCTCTGCCTCTGAGAACCGACTTCAGC (Hu STING(R284M/V147L/N154S/V155M); noepitope tag; nucleotide sequence) 149TGATAATAGGCTGGAGCCTCGGTGGCCTAGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCCAAACACCATTGTCACACTCCAGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC(3′ UTR used in STING V155M construct, containing miR122 binding site)150ATGGAGACCCCCAAGCCTAGAATCCTGCCCTGGCTGGTGAGCCAGCTGGACCTGGGCCAGCTGGAGGGCGTAGCCTGGCTGGACGAGAGCAGAACCAGATTCAGAATCCCCTGGAAGCACGGCCTGAGACAAGACGCCCAGATGGCCGACTTCGGCATCTTCCAGGCCTGGGCCGAGGCCAGCGGCGCCTACACCCCTGGCAAGGATAAGCCCGATGTGAGCACCTGGAAGAGAAACTTCAGAAGCGCCCTGAACAGAAAGGAGGTGCTGAGACTGGCCGCCGACAATAGCAAGGACCCCTACGACCCCCACAAGGTGTACGAGTTCGTTACCCCCGGCGCCAGGGACTTCGTGCACCTGGGCGCCAGCCCCGACACCAACGGCAAGAGCAGCCTGCCCCACAGCCAGGAGAACCTGCCCAAGCTGTTCGATGGCCTGATCCTGGGCCCCCTGAAGGACGAGGGCAGCAGCGACCTGGCCATCGTGAGCGACCCTAGCCAGCAGCTGCCCTCCCCCAACGTGAACAACTTCCTGAACCCCGCCCCCCAGGAGAACCCCCTGAAGCAACTGCTGGCCGAGGAGCAGTGGGAGTTCGAGGTGACCGCCTTCTACAGAGGCAGACAGGTGTTCCAGCAGACCCTGTTCTGCCCCGGCGGCCTGAGACTGGTAGGCAGCACCGCTGACATGACCCTGCCCTGGCAGCCCGTGACCCTGCCCGACCCCGAAGGCTTTCTGACCGACAAGCTGGTGAAGGAGTACGTCGGCCAAGTGCTGAAGGGCCTGGGCAACGGCCTGGCCCTGTGGCAGGCCGGCCAGTGCCTGTGGGCCCAGAGACTCGGCCACAGCCACGCCTTCTGGGCCCTGGGCGAGGAACTCCTGCCCGATAGCGGCAGAGGCCCCGACGGCGAGGTGCACAAGGACAAGGACGGCGCCGTGTTCGACCTGCGCCCCTTCGTGGCCGACCTGATCGCCTTCATGGAGGGCAGCGGCCACAGCCCCAGATATACCCTGTGGTTCTGCATGGGCGAGATGTGGCCCCAGGACCAGCCCTGGGTGAAGAGACTGGTGATGGTGAAGGTGGTGCCCACCTGCCTGAAAGAGCTGCTGGAGATGGCCAGAGAGGGCGGCGCCAGCTCCCTGAAAACCGTGGACCTGCACATTGACAACAGCCAGCCCATCAGCCTGACCAGCGACCAGTACAAGGCCTACCTGCAGGACCTGGTGGAGGACATGGACTTCCAGGCCACCGGCAACATC (super mouse IRF3 S396D; no epitope tag) 151ATGGGCACCCCCAAGCCCAGAATCCTGCCCTGGCTGGTGAGCCAGCTGGACCTGGGCCAGCTGGAGGGAGTGGCCTGGGTGAACAAGAGCAGAACCAGATTCAGAATCCCCTGGAAGCACGGCCTCAGACAGGACGCCCAGCAGGAGGACTTCGGCATTTTTCAGGCTTGGGCCGAGGCCACCGGCGCCTACGTGCCCGGCAGAGACAAGCCCGACCTGCCCACCTGGAAAAGAAACTTCAGAAGCGCCTTGAATAGAAAGGAGGGCCTGAGACTGGCCGAGGACAGAAGCAAGGACCCCCACGACCCTCACAAGATCTACGAGTTCGTGAATAGCGGCGTGGGCGACTTTAGCCAGCCCGACACCAGCCCCGACACCAACGGCGGCGGCAGCACCAGCGACACGCAGGAGGACATCCTGGATGAACTGCTGGGCAACATGGTGCTGGCCCCCCTGCCCGATCCCGGCCCCCCTTCGCTTGCCGTGGCCCCCGAGCCCTGCCCCCAGCCCCTGAGAAGCCCCTCTCTGGATAACCCCACCCCCTTCCCCAACCTGGGCCCCAGCGAGAATCCACTGAAGAGACTTCTGGTCCCCGGCGAGGAGTGGGAGTTCGAGGTGACCGCCTTCTACAGAGGCAGACAGGTGTTCCAGCAGACCATCAGCTGCCCCGAAGGCCTGAGATTAGTGGGCAGCGAAGTGGGCGACAGGACCCTGCCCGGGTGGCCCGTGACCCTGCCCGATCCCGGCATGAGCCTGACCGACAGAGGTGTGATGAGCTACGTGAGACACGTGCTGAGCTGCCTGGGCGGCGGCCTGGCACTGTGGAGAGCCGGCCAGTGGCTGTGGGCCCAGAGACTGGGCCACTGCCACACCTACTGGGCCGTGAGCGAGGAGCTGCTGCCCAACAGCGGCCACGGCCCCGACGGCGAGGTGCCCAAGGACAAGGAAGGGGGCGTGTTCGACCTGGGCCCCTTCATCGTAGACCTGATCACCTTTACCGAGGGCAGCGGCAGGAGCCCCAGATACGCCCTGTGGTTCTGCGTGGGCGAAAGCTGGCCCCAGGACCAGCCCTGGACCAAGAGACTGGTGATGGTGAAGGTAGTGCCCACCTGCCTGAGAGCCTTAGTGGAGATGGCCAGAGTGGGCGGGGCCAGCAGCCTGGAGAACACCGTGGATCTTCACATCGACAACAGCCACCCCCTGAGCCTGACCAGCGACCAGTACAAGGCCTACCTGCAGGACCTGGTGGAGGGCATGGACTTCCAGGGCCCCGGCGAGACC (super human IRF3 S396D; no epitopetag) 152ATGGCGCTGGCCCCCGAAAGAGCCGCCCCCAGAGTCCTCTTCGGCGAATGGCTCCTTGGCGAAATTTCGTCGGGCTGCTACGAGGGCTTACAATGGCTGGATGAGGCGAGAACCTGTTTCAGGGTGCCCTGGAAACACTTCGCCAGAAAGGATCTAAGCGAAGCAGATGCTAGAATTTTTAAGGCTTGGGCCGTGGCCAGGGGAAGATGGCCCCCCTCGAGCAGAGGCGGCGGCCCTCCCCCCGAGGCAGAAACGGCCGAGAGAGCCGGATGGAAAACCAATTTCAGATGCGCCCTGAGATCTACAAGAAGATTCGTGATGCTTAGAGACAACAGCGGAGATCCCGCCGATCCCCATAAGGTGTATGCCCTGTCCCGGGAGCTGTGCTGGAGGGAAGGGCCTGGCACTGACCAGACCGAAGCCGAAGCCCCCGCGGCCGTGCCGCCGCCCCAAGGAGGCCCACCAGGCCCTTTCCTCGCTCACACCCACGCCGGTCTGCAAGCCCCGGGACCTCTACCTGCCCCTGCCGGCGATAAAGGCGACCTGTTGCTGCAGGCCGTCCAACAGAGCTGCCTGGCCGATCATCTGCTCACAGCCAGCTGGGGCGCTGACCCCGTCCCAACAAAGGCCCCCGGTGAGGGCCAAGAAGGCCTGCCTCTGACCGGCGCCTGTGCCGGCGGCCCTGGCCTGCCTGCTGGCGAGCTGTACGGATGGGCTGTCGAAACCACTCCCTCCCCCGGCCCCCAACCTGCGGCCCTGACAACCGGCGAGGCAGCCGCACCCGAAAGCCCCCACCAGGCCGAACCCTACCTCAGTCCCAGCCCCTCCGCCTGCACCGCTGTGCAGGAGCCCAGCCCCGGTGCTCTGGACGTAACAATCATGTACAAAGGCAGAACCGTGCTTCAGAAGGTGGTTGGACACCCCTCCTGTACTTTTCTCTACGGCCCCCCCGACCCTGCCGTGAGAGCTACCGACCCGCAACAGGTGGCCTTTCCCTCGCCCGCCGAACTGCCCGATCAAAAACAGCTGAGATACACCGAGGAGCTGCTGAGACACGTGGCGCCGGGCTTACACCTAGAGTTGAGAGGCCCCCAACTCTGGGCCAGACGCATGGGCAAGTGTAAGGTGTACTGGGAGGTCGGGGGCCCTCCCGGCTCTGCCAGCCCCAGCACCCCTGCTTGTCTCTTGCCCAGAAACTGTGATACCCCCATCTTCGACTTCCGTGTATTTTTCCAGGAACTGGTCGAGTTTAGAGCCAGACAGAGACGAGGCAGCCCCAGATATACAATCTACCTCGGCTTCGGCCAGGACCTGAGTGCCGGCAGACCTAAGGAGAAGTCGCTGGTCCTAGTGAAGTTAGAGCCCTGGCTATGTAGAGTGCACCTGGAGGGCACCCAGAGAGAAGGAGTGAGCAGCCTGGACAGCAGCAGCCTGAGTCTGTGCCTGAGCTCCGCCAACTCGCTGTATGATGACATCGAGTGTTTCCTCATGGAGCTGGAGCAGCCCGCC (Wild-type Hu IRF7 isoform A; P037without epitope tag) 153ATGGCCCTTGCCCCTGAGCGGGCCGCCCCCAGAGTGTTATTCGGCGAGTGGCTGCTGGGCGAGATCAGCAGCGGCTGCTACGAGGGACTGCAGTGGCTGGACGAGGCTAGAACCTGCTTCAGAGTGCCCTGGAAGCATTTCGCCAGAAAAGACCTGAGCGAGGCTGATGCTAGAATCTTCAAAGCCTGGGCTGTGGCCCGAGGAAGATGGCCCCCCAGCAGCAGAGGAGGCGGCCCTCCTCCCGAGGCCGAAACCGCAGAGCGTGCTGGCTGGAAAACCAACTTTAGGTGTGCCCTGAGGAGCACCAGAAGATTCGTTATGCTCAGAGACAACAGCGGGGACCCCGCCGACCCGCACAAGGTGTACGCCTTAAGTAGGGAGCTGTGCTGGAGAGAGGGACCGGGGACCGACCAAACCGAGGCTGAGGCGCCCGCCGCCGTTCCACCTCCCCAGGGTGGTCCCCCAGGGCCCTTTCTGGCACACACCCACGCCGGATTACAGGCGCCAGGGCCCTTACCCGCCCCCGCCGGAGACAAAGGCGACCTCCTGCTGCAAGCCGTGCAACAAAGCTGCCTGGCCGATCACTTACTAACCGCTAGCTGGGGCGCCGATCCTGTTCCCACCAAGGCCCCCGGTGAAGGGCAAGAAGGACTGCCCTTAACCGGCGCCTGTGCCGGAGGCCCTGGTCTGCCAGCCGGCGAGCTGTACGGTTGGGCTGTCGAAACAACACCCAGTCCGGGCCCACAGCCTGCCGCTCTGACCACCGGCGAAGCCGCCGCCCCCGAGAGCCCACACCAGGCTGAACCCTACCTGAGCCCCAGCCCCAGCGCCTGCACCGCTGTGCAGGAGCCTAGCCCCGGCGCTCTTGATGTGACAATAATGTACAAGGGCAGGACCGTGCTGCAAAAGGTCGTGGGCCATCCGTCGTGTACCTTTCTGTACGGCCCTCCAGACCCCGCGGTTAGAGCCACCGACCCCCAGCAAGTCGCCTTCCCCTCCCCCGCCGAACTGCCCGACCAAAAGCAGCTGCGGTACACAGAAGAACTACTTAGACACGTGGCCCCCGGTCTGCACTTGGAGCTGAGAGGCCCCCAGCTCTGGGCCAGAAGAATGGGCAAGTGCAAAGTGTACTGGGAGGTGGGCGGCCCACCCGGCTCAGCTTCGCCCTCCACACCCGCATGCCTGCTGCCCAGAAATTGCGACACGCCCATCTTCGATTTTAGAGTGTTCTTTCAGGAGTTGGTGGAGTTCAGAGCCAGACAAAGACGCGGCAGCCCCAGATACACCATTTACCTCGGCTTCGGCCAGGACCTCAGCGCTGGCAGACCCAAGGAGAAGAGTCTGGTCCTCGTGAAGCTGGAGCCCTGGCTGTGCAGAGTGCACCTGGAGGGCACCCAGCGTGAAGGCGTGAGCAGCCTGGATTCAAGCGACCTGGACCTATGCCTAAGCAGCGCTAACTCACTGTACGACGATATCGAATGCTTCCTGATGGAACTGGAGCAGCCTGCC (constitutively active Hu IRF7S477D/S479D; P033 without epitope tag) 154ATGGCCCTGGCACCCGAGAGGGCCGCCCCCAGGGTGCTCTTCGGCGAGTGGTTACTAGGCGAAATTAGCAGCGGCTGCTATGAAGGCCTTCAGTGGCTGGACGAGGCCAGAACCTGCTTTAGAGTTCCCTGGAAGCACTTCGCCCGGAAAGATCTCTCTGAAGCCGACGCCAGAATATTCAAGGCCTGGGCTGTCGCCAGGGGCAGGTGGCCACCCTCCAGCCGAGGTGGCGGCCCTCCCCCTGAGGCTGAGACTGCGGAAAGGGCGGGCTGGAAGACCAATTTCAGATGCGCTCTGAGAAGCACCAGACGTTTTGTGATGCTAAGAGACAATAGCGGCGATCCCGCCGACCCCCATAAGGTATACGCACTGAGCCGAGAGCTCTGTTGGAGAGAAGGCCCCGGCACCGACCAGACCGAGGCTGAAGCCCCTGCAGCCGTGCCCCCCCCTCAAGGCGGGCCCCCCGGCCCCTTCCTGGCCCATACCCATGCAGGGTTACAAGCACCCGGGCCCTTGCCCGCCCCAGCGGGAGACAAGGGCGACCTCTTACTGCAGGCCGTGCAACAAAGTTGTCTGGCGGACCACCTGCTGACCGCATCATGGGGCGCGGATCCTGTGCCCACCAAGGCACCCGGCGAAGGCCAGGAGGGCCTGCCCTTGACCGGCGCCTGCGCTGGCGGACCCGGCCTACCTGCTGGCGAACTGTATGGCTGGGCCGTAGAGACGACTCCCAGCCCTGGCCCACAACCCGCGGCTTTGACCACCGGCGAAGCCGCCGCCCCCGAGTCTCCGCACCAGGCCGAGCCTTACCTCAGCCCAAGCCCTAGCGCCTGCACCGCCGTGCAAGAACCTAGCCCCGGAGCCCTGGATGTGACAATCATGTACAAGGGTAGAACCGTACTGCAAAAGGTGGTGGGTCATCCCAGCTGCACCTTTCTTTACGGCCCACCCGACCCTGCCGTGCGAGCCACAGACCCACAACAGGTCGCCTTCCCAAGCCCCGCCGAACTGCCCGATCAGAAACAGCTGAGATATACAGAGGAGCTTCTGCGGCACGTAGCTCCCGGCCTACATCTCGAGCTGAGGGGCCCACAACTGTGGGCCAGACGCATGGGCAAATGCAAGGTCTACTGGGAAGTGGGAGGCCCCCCCGGCAGCGCATCTCCCAGCACGCCCGCGTGCCTGCTGCCTAGAAATTGCGACACCCCCATCTTTGACTTCCGGGTATTCTTTCAGGAGCTGGTAGAGTTCAGAGCCAGGCAGCGGAGGGGCTCCCCCAGATACACAATCTACCTGGGCTTCGGACAGGACCTGTCCGCCGGCCGCCCCAAGGAAAAGAGCCTGGTGCTGGTGAAGCTGGAGCCCTGGCTGTGTAGGGTACACCTCGAAGGCACCCAGAGAGAAGGAGTGAGCTCGCTTGATGACAGCGATCTGTCGGATTGCCTTAGCAGCGCCAACAGCCTGTATGATGATATCGAGTGCTTCCTTATGGAACTGGAGCAGCCCGCC (constitutively active Hu IRF7S475D/S477D/L480D; P034 without epitope tag) 155ATGGCCCTAGCCCCCGAAAGAGCAGCTCCCAGAGTGCTGTTCGGCGAATGGCTGCTTGGCGAGATCAGCAGCGGCTGCTACGAAGGCCTGCAGTGGCTGGACGAAGCCCGCACCTGTTTCAGAGTGCCCTGGAAGCACTTCGCTAGAAAGGATTTGAGCGAGGCTGATGCTAGAATCTTTAAGGCTTGGGCTGTGGCAAGAGGCAGATGGCCGCCTAGTAGCAGAGGGGGCGGACCTCCCCCCGAGGCTGAGACCGCTGAGAGAGCAGGGTGGAAAACCAACTTCAGATGCGCGCTGAGAAGCACCCGAAGATTCGTGATGCTACGTGACAATAGCGGCGACCCCGCCGACCCCCACAAAGTGTACGCCCTGTCCCGAGAACTTTGCTGGAGAGAGGGACCCGGCACCGATCAAACAGAGGCTGAGGCCCCGGCCGCTGTACCCCCGCCCCAAGGAGGCCCCCCAGGCCCCTTTCTGGCTCATACACATGCCGGCCTGCAGGCACCCGGGCCCCTCCCGGCTCCTGCCGGCGACAAGGGCGATCTCCTTCTCCAGGCCGTGCAGCAGAGCTGCCTGGCCGATCACCTGCTGACCGCCTCGTGGGGCGCCGACCCCGTGCCCACCAAAGCCCCGGGTGAAGGCCAAGAGGGGCTCCCTTTAACCGGAGCATGCGCCGGAGGCCCCGGCCTGCCAGCCGGCGAGTTATATGGCTGGGCTGTGGAGACCACACCCTCCCCCGGCCCTCAACCCGCTGCCCTGACCACCGGTGAGGCCGCCGCCCCCGAGAGCCCACACCAGGCCGAACCCTACCTGAGCCCTAGCCCTAGCGCCTGCACCGCCGTGCAAGAACCCAGCCCCGGAGCCCTGGATGTGACCATTATGTACAAGGGCCGGACAGTGCTGCAAAAGGTTGTGGGACACCCGAGCTGCACCTTTCTGTACGGTCCGCCTGACCCCGCCGTGAGAGCCACGGACCCGCAGCAGGTGGCCTTCCCCTCACCCGCGGAGCTGCCCGACCAAAAGCAACTCAGATACACAGAAGAACTATTGCGTCACGTCGCGCCCGGCCTGCATCTGGAGCTGAGAGGCCCCCAGCTCTGGGCCAGAAGGATGGGCAAATGCAAGGTGTACTGGGAGGTGGGAGGCCCCCCCGGCAGCGCCAGCCCCAGCACTCCCGCGTGCCTGCTGCCCAGAAATTGCGACACTCCCATCTTCGATTTCAGGGTGTTCTTCCAGGAGCTGGTGGAGTTCAGAGCCAGGCAGAGAAGGGGTAGCCCCAGATACACAATCTATCTAGGCTTTGGACAAGATCTGAGCGCCGGCCGGCCTAAGGAAAAAAGCCTGGTGCTGGTAAAGCTGGAGCCGTGGCTTTGTAGAGTGCACCTGGAGGGGACGCAGCGAGAGGGCGTGAGCAGCTTAGACGACGATGACTTGGATCTGTGTCTCGACAGCGCCAACGACTTGTACGACGACATCGAGTGCTTCCTGATGGAACTGGAGCAGCCCGCC (constitutively active Hu IRF7S475D/S476D/S477D/S479D/S483D/S487D; P035 without epitope tag) 156ATGGCCCTGGCCCCCGAGAGAGCCGCCCCCAGAGTGCTCTTCGGCGAGTGGCTGCTGGGCGAGATAAGCAGCGGCTGCTACGAAGGTCTGCAGTGGCTAGACGAGGCCAGAACCTGCTTTAGAGTGCCCTGGAAGCACTTCGCTCGAAAGGACCTGTCCGAGGCCGATGCTAGAATTTTTAAGGCTTGGGCCGTCGCTAGGGGAAGATGGCCCCCTAGCAGTAGAGGCGGCGGCCCCCCTCCCGAAGCCGAGACGGCCGAGAGGGCCGGCTGGAAAACCAATTTCAGATGCGCCCTGAGGAGCACCCGCAGGTTCGTAATGCTGCGAGACAATAGCGGCGATCCTGCGGATCCTCACAAGGTTTACGCCTTGAGTAGAGAACTGTGCTGGCGGGAGGGCCCCGGAACCGACCAGACGGAGGCAGAGGCACCCGCTGCCGTGCCCCCCCCTCAAGGAGGACCCCCTGGACCCTTTCTGGCCCACACCCACGCTGGTCTGCAGGCCCCAGGCCCACTGCCCGCCCCAGCGGGCGATAAGGGTGACCTGCTCCTACAGGCGGTGCAACAGAGCTGTCTGGCCGACCACCTGTTGACCGCCAGCTGGGGGGCCGACCCGGTGCCCACCAAAGCTCCCGGAGAGGGCCAAGAAGGCCTCCCACTAACTGGCGCCTGCGCCGGGGGCCCGGGATTACCCGCCGGCGAGCTGTATGGCTGGGCCGTGGAGACCACGCCCAGCCCCGAGGGCGTGTCGTCCCTGGACAGCAGCAGCCTGAGCCTGTGCCTGAGCTCCGCCAACAGCCTGTATGACGACATCGAGTGCTTCCTGATGGAGCTGGAACAACCCGCC (constitutively active truncatedHu IRF7 1-246 + 468-503; P032 without epitope tag) 157ATGGCACTGGCGCCTGAAAGAGCCGCTCCGCGTGTGCTCTTCGGCGAGTGGCTGCTGGGCGAGATCAGCTCCGGCTGCTACGAGGGTCTACAGTGGCTGGACGAGGCCAGAACCTGTTTTAGAGTGCCCTGGAAGCACTTCGCGAGAAAGGACCTGAGCGAGGCCGACGCCAGAATCTTCAAAGCCTGGGCAGTGGCTAGGGGCAGATGGCCTCCCAGCAGCCGGGGCGGCGGCCCACCCCCCGAGGCCGAAACCGCCGAAAGAGCTGGCTGGAAGACCAACTTCAGATGCGCCCTGAGAAGCACCAGAAGATTTGTCATGCTGAGAGATAATTCAGGAGACCCCGCCGACCCTCACAAGGTGTACGCCCTGTCCAGAGAGCTGTGTTGGAGAGAGGGCCCCGGAACCGACCAGACCGAGGCCGAGGCTCCAGCTGCCGTGCCACCCCCCCAAGGCGGACCACCCGGCCCCTTCTTGGCACATACGCACGCCGGCCTCCAGGCTCCCGGCCCTCTGCCCGCCCCTGCTGGTGACAAAGGCGATCTGCTGCTGCAAGCCGTCCAGCAATCCTGCTTGGCTGACCACCTGCTGACCGCTAGCTGGGGAGCCGACCCCGTTCCCACCAAGGCTCCCGGAGAAGGACAGGAGGGCCTGCCCCTTACCGGCGCTTGCGCGGGGGGCCCTGGCTTGCCTGCCGGCGAACTGTACGGCTGGGCCGTGGAGACCACGCCTTCCCCCGAGGGCGTGTCCAGCCTGGACGATGATGACCTGGATCTGTGCCTGGACAGCGCCAACGACCTGTACGATGACATCGAGTGCTTTTTGATGGAGCTGGAGCAGCCCGCC (constitutively active truncatedHu IRF7 1-246 + 468-503 plus S475D/S476D/S477D/S479D/S483D/S487D; P036without epitope tag) 158ATGGCCCTGGCCCCCGAGAGAGCCGCGCCCAGAGTGCTGTTCGGCGAATGGCTGCTGGGCGAGATCAGCAGCGGCTGCTATGAGGGCCTGCAGTGGCTCGACGAAGCCAGGACGTGCTTCAGAGTCCCCTGGAAGCACTTCGCCAGAAAGGATCTGAGCGAGGCTGACGCCAGAATCTTCAAGGCCTGGGCAGTTGCGCGTGGGAGATGGCCCCCCAGCTCGCGGGGCGGCGGTCCCCCCCCTGAGGCCGAGACCGCCGAAAGAGCCGGATGGAAAACCAACTTTCGATGCGCCCTCAGAAGCACCAGACGGTTTGTGATGCTGAGAGATAACAGCGGCGACCCTGCAGACCCCCATAAAGTGTATGCCCTGAGCAGAGAGCTGTGTTGGCGAGAGGGCCCCGGAACCGACCAAACCGAGGCCGAGGCCCCCGCCGCCGTACCCCCCCCTCAAGGCCCCCAGCCTGCTGCTCTGACCACGGGAGAAGCCGCCGCTCCTGAGAGCCCCCACCAAGCCGAGCCCTATCTGAGCCCTAGCCCCAGCGCCTGCACCGCCGTGCAGGAGCCCTCACCGGGCGCCCTAGACGTGACCATCATGTACAAGGGGCGCACGGTGCTGCAAAAGGTGGTGGGCCACCCCAGCTGCACCTTCCTGTACGGCCCCCCCGACCCTGCCGTGAGAGCCACCGACCCCCAGCAAGTCGCCTTCCCCAGCCCCGCCGAGCTGCCCGACCAGAAGCAGCTGAGGTACACCGAGGAGTTGCTGAGACATGTGGCCCCCGGCTTGCACCTCGAGCTGAGAGGCCCGCAGCTCTGGGCCAGAAGAATGGGCAAGTGCAAGGTGTACTGGGAGGTGGGCGGCCCCCCCGGCAGCGCGAGCCCAAGCACCCCGGCCTGCCTGCTGCCTAGAAACTGCGACACCCCTATCTTCGACTTCAGAGTATTTTTCCAGGAGCTGGTCGAGTTCAGGGCCAGACAGCGTAGAGGCAGCCCCAGATACACCATCTACCTTGGATTCGGCCAGGACCTGAGCGCCGGCAGACCCAAAGAGAAGTCCCTGGTACTGGTGAAGCTAGAGCCCTGGCTGTGTAGGGTGCATCTGGAAGGCACCCAAAGAGAGGGCGTAAGCTCGCTTGACAGCAGCAGCCTCAGCCTGTGCCTGAGCAGCGCTAACAGCTTATACGACGACATCGAGTGCTTCCTGATGGAGCTGGAACAACCCGCC (truncated Hu IRF71-151 + 247-503; P038 without epitope tag; null mutation) 159ATGGGCGGCCCTCCCGGGCCTTTCCTGGCCCATACACACGCCGGCCTACAGGCTCCTGGCCCTCTGCCCGCCCCGGCCGGCGACAAGGGCGACCTCCTGCTGCAGGCCGTGCAGCAGTCCTGTCTGGCCGACCACCTGCTGACTGCTAGCTGGGGCGCCGATCCCGTGCCCACCAAGGCCCCAGGAGAGGGGCAAGAGGGCCTGCCTCTAACCGGCGCATGCGCAGGTGGACCAGGCCTCCCCGCCGGCGAGCTGTATGGTTGGGCCGTGGAGACAACCCCCAGCCCCGGCCCGCAGCCTGCTGCGCTGACCACAGGCGAGGCCGCTGCCCCTGAGAGCCCCCACCAAGCTGAACCCTACCTGAGCCCCAGCCCCTCTGCCTGCACAGCGGTGCAGGAGCCCAGTCCCGGCGCCTTGGACGTGACCATCATGTATAAGGGCAGGACTGTGTTACAAAAGGTAGTGGGCCACCCAAGTTGTACCTTTCTGTACGGGCCCCCCGACCCAGCCGTGCGCGCCACCGACCCCCAGCAGGTGGCCTTCCCCAGCCCCGCTGAGTTGCCCGATCAGAAACAACTCCGGTACACCGAGGAATTACTTAGACATGTGGCTCCCGGCCTGCATCTGGAGCTTAGAGGTCCACAGTTGTGGGCCAGAAGAATGGGCAAGTGCAAGGTTTATTGGGAGGTCGGAGGCCCCCCGGGCAGCGCCAGCCCCAGCACCCCCGCCTGTCTTCTGCCCAGAAACTGCGACACCCCAATCTTCGATTTCAGAGTGTTTTTCCAGGAACTGGTGGAGTTCAGAGCAAGGCAAAGAAGAGGCAGCCCTAGATACACCATCTACCTGGGCTTTGGCCAAGACCTGAGCGCCGGCAGACCCAAGGAAAAATCCCTGGTCCTGGTGAAACTGGAGCCCTGGCTGTGCAGAGTCCACCTGGAGGGCACCCAGAGAGAGGGCGTGAGCAGCCTGGACTCGAGCAGCCTGTCCCTGTGTCTGAGCAGCGCGAATTCGCTATATGACGACATCGAATGCTTTCTGATGGAGCTGGAACAGCCCGCC (truncated Hu IRF7 152-503; P039 withoutepitope tag; null mutation) 160ATGCCTCACAGCAGCCTCCACCCTAGCATCCCTTGCCCTAGAGGCCACGGCGCCCAGAAGGCCGCCCTCGTGCTTTTAAGCGCCTGCTTGGTGACCCTTTGGGGCTTGGGCGAGCCTCCAGAGCACACCTTGAGATATTTGGTGCTCCACCTGGCCAGCCTTCAGCTGGGCTTGTTACTCAACGGCGTGTGCAGCCTGGCCGAGGAGCTGAGACACATCCACAGCAGATACAGAGGCAGCTACTGGAGAACCGTGAGAGCGTGTCTGGGCTGCCCTCTGAGAAGAGGCGCCTTGCTTCTTCTCAGTATCTACTTCTACTACTCCCTGCCTAACGCCGTGGGCCCTCCTTTCACCTGGATGCTGGCACTGCTCGGCCTCAGCCAGGCCCTGAACATCTTGTTGGGCTTGAAGGGCCTGGCCCCTGCCGAGATCAGCGCCGTGTGCGAGAAGGGCAACTTCAACATGGCCCACGGATTGGCTTGGAGCTACTACATCGGCTACCTGAGACTGATCCTGCCTGAGCTGCAGGCCAGAATCAGAACCTACAACCAGCACTACAACAACCTGCTGCGCGGCGCAGTGAGCCAGAGACTGTATATTCTGCTGCCTCTGGACTGCGGCGTGCCTGACAACCTGAGCATGGCCGACCCTAACATCAGATTCCTGGACAAGCTGCCTCAGCAGACCGGCGACCACGCCGGCATCAAGGACAGAGTGTACAGCAACAGCATCTATGAGCTGCTCGAGAATGGCCAGAGAGCCGGCACCTGCGTGCTGGAGTACGCCACCCCTCTGCAGACCCTGTTCGCCATGAGCCAGTATAGTCAAGCTGGCTTCAGCAGAGAGGACAGACTGGAGCAGGCCAAGCTGTTCTGCAGAACCCTGGAGGACATTCTGGCTGACGCCCCTGAGAGCCAGAACAACTGCCGACTGATCGCCTACCAGGAACCAGCCGACGACAGCAGCTTCAGTCTTTCTCAGGAGGTTCTTCGCCACTTGCGCCAGGAGGAGAAGGAGGAGGTGACCGTGGGCAGCCTGAAGACCTCCGCAGTCCCTAGCACCAGCACCATGAGTCAGGAGCCGGAGCTATTAATCAGCGGCATGGAGAAGCCTCTTCCACTCCGAACCGACTTCAGCGCCACCAACTTCAGCCTGCTGAAGCAGGCAGGTGACGTTGAGGAGAATCCGGGACCTATGACCGAGTACAAGCTGGTGGTTGTGGGCGCCGACGGCGTGGGCAAGAGCGCCCTGACCATCCAGCTGATCCAG (KRAS(G12D)25mer_nt.STING(V155M)) 161ATGACCGAGTACAAGCTAGTAGTCGTGGGCGCCGACGGCGTGGGCAAGAGCGCCCTCACCATCCAGCTAATCCAGGCCACCAACTTCAGCTTGCTCAAGCAGGCCGGCGACGTGGAGGAGAACCCAGGCCCTATGCCTCACAGCAGCCTTCACCCTAGCATCCCTTGCCCTAGAGGCCACGGCGCCCAGAAGGCCGCCCTGGTGCTGCTGAGCGCCTGCCTGGTGACCCTGTGGGGCCTGGGCGAGCCTCCTGAGCACACCCTGAGATATCTGGTGCTTCACCTGGCCAGTTTACAGCTGGGCCTGCTTCTTAACGGCGTGTGCAGCCTGGCCGAGGAGCTGAGACACATCCACAGCAGATACAGAGGCAGCTACTGGAGAACCGTGAGAGCCTGCCTAGGCTGCCCTCTGAGAAGAGGCGCTCTGTTGCTACTTTCCATCTACTTCTACTACTCCCTGCCTAACGCCGTGGGCCCTCCTTTCACTTGGATGCTGGCGTTGCTGGGTCTGAGCCAGGCCCTGAACATCCTTCTCGGTCTGAAGGGCCTGGCCCCTGCCGAGATCAGCGCCGTGTGCGAGAAGGGCAACTTCAACATGGCCCACGGACTCGCCTGGAGCTACTACATCGGCTACCTGAGACTGATCCTGCCTGAGCTGCAGGCCAGAATCAGAACCTACAACCAGCACTACAACAACCTGCTGCGGGGCGCCGTGAGCCAGAGACTGTATATACTTCTTCCTCTGGACTGCGGCGTGCCTGACAACCTGAGCATGGCCGACCCTAACATCAGATTCCTGGACAAGCTGCCTCAGCAGACCGGCGACCACGCCGGCATCAAGGACAGAGTGTACAGCAACTCCATTTATGAGCTGCTCGAGAATGGCCAGAGAGCCGGCACCTGCGTGCTGGAGTACGCCACCCCTCTGCAGACCCTGTTCGCCATGAGCCAGTACAGTCAGGCTGGATTCAGCAGAGAGGACAGACTGGAGCAGGCCAAGCTGTTCTGCAGGACACTGGAGGACATACTAGCAGACGCCCCTGAGAGCCAGAACAACTGCAGACTGATTGCCTACCAGGAGCCTGCGGACGACAGCTCCTTCAGTCTGAGTCAGGAGGTGTTGCGGCACTTACGCCAAGAAGAGAAGGAGGAGGTGACCGTGGGCAGCCTGAAGACTAGCGCTGTGCCTAGCACCAGCACAATGTCACAGGAGCCGGAATTGCTAATCAGCGGCATGGAGAAGCCTCTCCCATTACGTACCGACTTCAGC (KRAS(G12D)25mer_ct.STING(V155M)) 162ATGCCTCACAGCAGCCTTCACCCTAGCATCCCTTGCCCTAGAGGCCACGGCGCCCAGAAGGCCGCCCTAGTGCTCCTTAGCGCCTGCCTCGTGACCCTATGGGGCTTAGGCGAGCCTCCAGAGCACACCTTGAGATACCTCGTCCTCCACCTGGCTAGTCTACAGCTGGGCCTTCTCCTCAACGGCGTGTGCAGCCTGGCCGAGGAGCTGAGACACATCCACAGCAGATACAGAGGCAGCTACTGGAGAACCGTGAGAGCGTGCCTGGGCTGCCCTCTGAGAAGAGGCGCACTGCTGTTACTCAGCATCTACTTCTACTACTCACTGCCAAACGCCGTGGGCCCTCCTTTCACCTGGATGCTGGCCTTGCTCGGATTGAGCCAGGCCCTGAACATTTTACTGGGATTGAAGGGCCTGGCCCCTGCCGAGATCAGCGCCGTGTGCGAGAAGGGCAACTTCAACATGGCCCACGGCCTAGCTTGGAGCTACTACATCGGCTACCTGAGACTGATCCTGCCTGAGCTGCAGGCCAGAATCAGAACCTACAACCAGCACTACAACAACCTGCTGCGTGGAGCGGTGAGCCAGAGACTGTATATCCTCCTGCCTCTGGACTGCGGAGTGCCTGACAACCTGAGCATGGCCGACCCTAACATCAGATTCCTGGACAAGCTGCCTCAGCAGACCGGCGACCACGCCGGCATCAAGGACAGAGTGTACAGCAACTCAATCTACGAGCTGTTGGAGAATGGCCAGAGAGCCGGCACCTGCGTGCTGGAGTACGCCACCCCTCTGCAGACCCTGTTCGCCATGAGCCAGTACTCTCAGGCAGGCTTCAGCAGAGAGGACAGACTGGAGCAGGCCAAGCTGTTCTGCAGAACCCTGGAGGACATCCTGGCGGACGCCCCTGAGAGCCAGAACAACTGCCGGCTTATCGCCTACCAGGAGCCAGCAGACGACAGCAGCTTCTCTCTCTCACAAGAGGTACTGCGCCATCTTCGCCAGGAGGAGAAGGAGGAGGTGACCGTGGGCAGCCTGAAGACATCCGCCGTACCTAGCACCAGCACCATGTCTCAGGAACCGGAACTGTTGATCAGCGGCATGGAGAAGCCTCTGCCACTGCGCACCGACTTCAGCGCCACCAACTTCTCCCTACTGAAGCAAGCCGGTGACGTTGAAGAGAACCCTGGCCCTATGACCGAGTACAAGCTGGTAGTAGTAGGCGCCGACGGCGTGGGCAAGAGCGCCCTGACCATCCAGCTGATCCAGATGACTGAATATAAGCTTGTCGTCGTGGGCGCAGATGGCGTTGGTAAGAGCGCACTTACAATTCAACTCATTCAGATGACGGAGTATAAGCTGGTGGTGGTCGGAGCTGACGGCGTAGGCAAGAGTGCCCTTACTATTCAGCTAATTCAG (KRAS(G12D)25mer^3_nt.STING(V155M)) 163ATGACCGAGTACAAGCTTGTGGTGGTTGGCGCCGACGGCGTGGGCAAGAGCGCCTTAACCATCCAGCTTATCCAGATGACAGAGTATAAGCTAGTGGTGGTCGGCGCAGACGGAGTGGGAAAGAGTGCATTAACTATTCAACTCATCCAAATGACCGAATACAAGCTAGTAGTTGTGGGTGCAGATGGCGTCGGCAAGTCTGCACTGACAATTCAGCTCATCCAGGCCACCAACTTCAGCCTGCTGAAGCAGGCCGGCGACGTGGAGGAGAACCCTGGCCCTATGCCTCACAGCAGCCTGCACCCTAGCATCCCTTGCCCTAGAGGCCACGGCGCCCAGAAGGCCGCCCTGGTGCTGCTGAGCGCCTGCCTGGTGACCCTGTGGGGCCTGGGCGAGCCTCCTGAGCACACCCTGAGATACCTAGTTTTGCACCTGGCTTCTCTGCAGCTGGGCCTACTGCTCAACGGCGTGTGCAGCCTGGCCGAGGAGCTGAGACACATCCACAGCAGATACAGAGGCAGCTACTGGAGAACCGTGAGAGCATGCTTAGGCTGCCCTCTGAGAAGAGGCGCTCTGCTCCTCTTGTCCATCTACTTCTACTACTCGCTACCTAACGCCGTGGGCCCTCCTTTCACCTGGATGCTGGCCCTCTTGGGATTAAGCCAGGCCCTGAACATCTTGCTGGGACTGAAGGGCCTGGCCCCTGCCGAGATCAGCGCCGTGTGCGAGAAGGGCAACTTCAACATGGCCCACGGACTCGCTTGGAGCTACTACATCGGCTACCTGAGACTGATCCTGCCTGAGCTGCAGGCCAGAATCAGAACCTACAACCAGCACTACAACAACCTGCTGCGGGGAGCAGTGAGCCAGAGACTGTATATTCTGCTCCCTCTGGACTGCGGCGTGCCTGACAACCTGAGCATGGCCGACCCTAACATCAGATTCCTGGACAAGCTGCCTCAGCAGACCGGCGACCACGCCGGCATCAAGGACAGAGTGTACAGCAACAGCATTTACGAGCTGCTGGAGAACGGCCAGAGAGCCGGCACCTGCGTGCTGGAGTACGCCACCCCTCTGCAGACCCTGTTCGCCATGAGCCAGTACTCCCAGGCAGGATTCAGCAGAGAGGACAGACTGGAGCAGGCCAAGCTGTTCTGCCGTACTCTTGAGGACATCCTTGCAGACGCCCCTGAGAGCCAGAACAACTGCCGGTTGATTGCCTACCAGGAACCGGCAGACGACAGCTCATTCTCCTTGTCTCAGGAGGTCCTTAGACACCTGCGGCAGGAGGAGAAGGAGGAGGTGACCGTGGGCAGCCTGAAGACATCCGCCGTGCCTAGCACGTCTACCATGTCCCAGGAGCCGGAACTGCTAATCAGCGGCATGGAGAAGCCTCTGCCTCTCAGGACCGACTTCAGC (KRAS(G12D)25mer^3_ct.STING(V155M)) 164MPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEELRHIHSRYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALNILLGLKGLAPAEISAVCEKGNFNVAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDHAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDkLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLRTDFST (HuSTING (R284K) var; no epitope tag) 165ATGCCCCATAGCAGCCTGCACCCCAGCATCCCCTGCCCCAGAGGCCACGGCGCCCAGAAGGCCGCCCTGGTCCTGCTGAGCGCATGCCTGGTCACCCTGTGGGGCCTGGGCGAGCCCCCCGAGCACACCCTGAGATACCTGGTGCTGCACCTCGCCAGCCTGCAGCTGGGCCTGCTGCTGAACGGCGTGTGCAGCCTGGCCGAGGAGCTGAGACACATCCACAGCAGATATAGAGGCAGCTACTGGAGAACCGTGAGAGCTTGCCTCGGCTGCCCCCTGAGAAGAGGCGCCCTGCTGCTGCTGAGCATCTACTTTTACTACAGCCTGCCCAACGCTGTGGGCCCCCCTTTCACGTGGATGCTCGCCCTGCTGGGACTGAGCCAGGCCCTGAACATCCTGCTGGGCCTTAAGGGCCTAGCCCCCGCCGAGATCAGCGCCGTGTGCGAGAAGGGCAACTTCAATGTGGCCCACGGCCTGGCCTGGAGCTACTACATCGGCTACCTGAGACTGATCCTGCCCGAGCTGCAGGCCAGAATCAGAACCTACAATCAGCACTACAACAACCTGCTGAGAGGCGCCGTGAGCCAGAGACTGTACATCCTGCTGCCCCTGGACTGCGGCGTGCCCGACAACCTCAGCATGGCCGACCCCAACATCAGATTCCTGGACAAGCTGCCCCAGCAGACCGGCGACCACGCCGGCATCAAGGATCGCGTGTACAGCAACAGCATCTACGAGCTGCTGGAAAACGGCCAGAGAGCCGGAACCTGCGTGCTGGAGTACGCCACACCCCTGCAGACCCTGTTCGCCATGAGCCAGTACAGCCAGGCCGGCTTCAGCAGAGAGGACAAGCTGGAGCAGGCCAAGCTGTTCTGCAGAACCCTGGAGGATATCCTCGCCGACGCCCCCGAGAGCCAGAACAACTGCAGGCTGATCGCGTACCAGGAGCCCGCTGACGACAGCAGCTTTAGCCTGAGCCAGGAGGTGCTGAGACATCTGCGTCAAGAGGAAAAGGAGGAGGTGACCGTGGGCTCCCTGAAGACCAGCGCCGTGCCCAGCACCAGCACCATGAGCCAGGAGCCCGAGCTGCTGATCAGCGGCATGGAGAAGCCACTGCCCCTCAGAACCGACTTCAGCACC (Hu STING (R284K) var; no epitopetag) 166MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQRVEDAFYTLVREIRQYRLKKISKEEKTPGCVKIKKC Human KRAS sp/P01116[1-186] 1675′^(7Me)G_(ppp)G_(2′OMe)GGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGACCGAGUACAAGCUCGUGGUCGUCGGCGCCGACGGGGUAGGCAAGUCCGCUCUGACCAUUCAGCUCAUCCAGAUGACGGAGUACAAACUCGUGGUAGUGGGAGCCGUGGGUGUGGGCAAGAGCGCGCUCACCAUCCAACUCAUCCAAAUGACCGAAUAUAAACUCGUCGUGGUGGGAGCCGGCGACGUGGGAAAGAGCGCCCUUACCAUCCAGUUAAUCCAGAUGACAGAAUACAAGCUGGUGGUGGUCGGUGCCUGCGGCGUGGGUAAGUCCGCCCUGACAAUCCAGCUGAUCCAGUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAUCUAG_(OH)3′ Where: A, C G& U = AMP, CMP, GMP & N1-ψUMP, respectively; Me = methyl; p = inorganicphosphate (KRAS concatemer mRNA sequence; CX-012908) 1685′^(7Me)G_(ppp)G_(2′OMe)GGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGCCCCACAGUAGCCUCCACCCCAGCAUCCCCUGCCCCAGAGGCCACGGCGCACAGAAGGCCGCCCUGGUGCUGCUGAGCGCCUGUCUGGUGACCCUGUGGGGUCUGGGCGAGCCCCCCGAGCACACCCUGCGGUACCUCGUGCUGCAUCUGGCCAGCCUGCAGCUGGGCCUGCUGCUGAACGGCGUGUGCAGCCUGGCCGAAGAGCUGAGACACAUCCACAGCAGAUACAGAGGCUCCUACUGGAGAACCGUCAGAGCCUGCCUCGGCUGUCCCCUGAGAAGAGGCGCCCUGCUGCUCCUGAGCAUCUACUUCUACUACAGCCUGCCCAACGCCGUGGGCCCCCCCUUCACCUGGAUGCUGGCCCUGCUGGGCCUGAGCCAGGCCCUGAACAUCCUGCUGGGCCUGAAGGGCUUGGCCCCCGCCGAGAUCUCCGCCGUGUGCGAGAAGGGCAACUUCAACAUGGCCCAUGGCCUUGCCUGGUCCUACUACAUCGGCUACCUGAGACUGAUCCUGCCCGAGCUGCAGGCCAGAAUCAGAACCUACAACCAGCACUACAACAACCUGCUGAGAGGCGCCGUGAGCCAAAGACUGUACAUCCUGCUGCCCCUGGACUGCGGCGUGCCCGACAACCUUAGCAUGGCCGACCCCAACAUCAGAUUCCUGGACAAGCUGCCCCAGCAGACCGGCGACCACGCCGGCAUCAAGGACAGAGUGUACAGCAACAGCAUCUACGAGCUGCUGGAGAACGGCCAGAGAGCCGGCACCUGCGUGCUGGAGUACGCCACCCCCCUGCAGACCCUGUUCGCCAUGAGCCAGUACAGCCAGGCCGGCUUCAGCAGAGAGGACAGACUGGAGCAAGCCAAGCUGUUCUGCAGAACCCUGGAGGACAUCCUGGCGGACGCCCCCGAGAGCCAAAACAACUGCAGACUGAUCGCCUACCAGGAGCCCGCCGACGACAGCAGCUUCAGCCUGAGCCAGGAAGUGCUGAGACACCUGAGACAGGAAGAGAAGGAGGAGGUGACCGUGGGAAGCCUGAAGACCAGCGCCGUGCCCAGCACCAGCACCAUGAGCCAGGAGCCCGAGCUGCUGAUCAGCGGCAUGGAGAAGCCCCUGCCCCUGAGAACCGACUUCAGCUGAUAAUAGGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCCAAACACCAUUGUCACACUCCAGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAUCUAG_(OH)3′ Where: A, C G & U = AMP, CMP, GMP &N1-ψUMP, respectively; Me = methyl; p = inorganic phosphate; underline= miR-122 binding site (STING mRNA sequence; CX-012871) 169AUGACCGAGUACAAGCUCGUGGUCGUCGGCGCCGACGGGGUAGGCAAGUCCGCUCUGACCAUUCAGCUCAUCCAGAUGACGGAGUACAAACUCGUGGUAGUGGGAGCCGUGGGUGUGGGCAAGAGCGCGCUCACCAUCCAACUCAUCCAAAUGACCGAAUAUAAACUCGUCGUGGUGGGAGCCGGCGACGUGGGAAAGAGCGCCCUUACCAUCCAGUUAAUCCAGAUGACAGAAUACAAGCUGGUGGUGGUCGGUGCCUGCGGCGUGGGUAAGUCCGCCCUGACAAUCCAGCUGAUCCAG (KRAS(G12D G12V G13D G12C) 100mer “4MUT” nt. seq)170AUGCCCCACAGUAGCCUCCACCCCAGCAUCCCCUGCCCCAGAGGCCACGGCGCACAGAAGGCCGCCCUGGUGCUGCUGAGCGCCUGUCUGGUGACCCUGUGGGGUCUGGGCGAGCCCCCCGAGCACACCCUGCGGUACCUCGUGCUGCAUCUGGCCAGCCUGCAGCUGGGCCUGCUGCUGAACGGCGUGUGCAGCCUGGCCGAAGAGCUGAGACACAUCCACAGCAGAUACAGAGGCUCCUACUGGAGAACCGUCAGAGCCUGCCUCGGCUGUCCCCUGAGAAGAGGCGCCCUGCUGCUCCUGAGCAUCUACUUCUACUACAGCCUGCCCAACGCCGUGGGCCCCCCCUUCACCUGGAUGCUGGCCCUGCUGGGCCUGAGCCAGGCCCUGAACAUCCUGCUGGGCCUGAAGGGCUUGGCCCCCGCCGAGAUCUCCGCCGUGUGCGAGAAGGGCAACUUCAACAUGGCCCAUGGCCUUGCCUGGUCCUACUACAUCGGCUACCUGAGACUGAUCCUGCCCGAGCUGCAGGCCAGAAUCAGAACCUACAACCAGCACUACAACAACCUGCUGAGAGGCGCCGUGAGCCAAAGACUGUACAUCCUGCUGCCCCUGGACUGCGGCGUGCCCGACAACCUUAGCAUGGCCGACCCCAACAUCAGAUUCCUGGACAAGCUGCCCCAGCAGACCGGCGACCACGCCGGCAUCAAGGACAGAGUGUACAGCAACAGCAUCUACGAGCUGCUGGAGAACGGCCAGAGAGCCGGCACCUGCGUGCUGGAGUACGCCACCCCCCUGCAGACCCUGUUCGCCAUGAGCCAGUACAGCCAGGCCGGCUUCAGCAGAGAGGACAGACUGGAGCAAGCCAAGCUGUUCUGCAGAACCCUGGAGGACAUCCUGGCGGACGCCCCCGAGAGCCAAAACAACUGCAGACUGAUCGCCUACCAGGAGCCCGCCGACGACAGCAGCUUCAGCCUGAGCCAGGAAGUGCUGAGACACCUGAGACAGGAAGAGAAGGAGGAGGUGACCGUGGGAAGCCUGAAGACCAGCGCCGUGCCCAGCACCAGCACCAUGAGCCAGGAGCCCGAGCUGCUGAUCAGCGGCAUGGAGAAGCCCCUGCCCCUGAGAACCGACUUCAGC (huSTING(V155M); no epitope tag; nucleotide sequence) 171CCUUAGCAGAGCUGUGGAGUGUGACAAUGGUGUUUGUGUCUAAACUAUCAAACGCCAUUAUCACACUAAAUAGCUACUGCUAGGC (mir-122) 172 AACGCCAUUAUCACACUAAAUA (mir-122-3p_(—)173 UAUUUAGUGUGAUAAUGGCGUU (mir-122-3p binding site) 174UGGAGUGUGACAAUGGUGUUUG (mir-122-5p) 175 CAAACACCAUUGUCACACUCCA(mir-122-5p binding site) 176GGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC (5′ UTR) 177 CCGCCGCCGCCG178 CCGCCGCCGCCGCCG 179 CCCCGGCGCC (V1) 180GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGA (5′UTR) 181GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGACCCCGGCGCCGCCACC (V1-UTR) 182GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGACCCCGGCGCCACC (V2-UTR) 183MKLVVVGACGVGKSAMKLVVVGACGVGKSAMKLVVVGACGVGKSA (KRAS G12C 15mer^3) 184ATGACCGAGTACAAGCTCGTGGTTGTTGGCGCCTGCGGCGTGGGCAAGAGCGCCCTCACCATCCAGCTCATCCAGATGACAGAGTATAAGTTAGTCGTTGTCGGAGCTTGCGGAGTTGGAAAGTCGGCGCTCACCATTCAACTCATACAAATGACAGAATATAAGTTAGTGGTGGTGGGTGCGTGTGGCGTTGGCAAGAGTGCGCTTACTATCCAGCTCATTCAG (KRAS G12C 25mer^3 nucleotide sequence) 185UCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC (5′ UTR) 186UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (3′ UTR) 187UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCCAAACACCAUUGUCACACUCCAUCCCCCCAGCCCCUCCUCCCCUUCCUCCAUAAAGUAGGAAACACUACAUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (3′ UTR with mi-122 and mi-142.3p sites) 188GGAAGCGGAGCUACUAACUUCAGCCUGCUGAAGCAGGCUGGAGACGUGGAGGAGAACCCUGGACCU(Nucleotide sequence encoding 2A peptide) 189UCCGGACUCAGAUCCGGGGAUCUCAAAAUUGUCGCUCCUGUCAAACAAACUCUUAACUUUGAUUUACUCAAACUGGCUGGGGAUGUAGAAAGCAAUCCAGGUCCACUC (Nucleotide sequence encoding 2Apeptide) 190AUGACCGAGUACAAGCUGGUGGUGGUGGGCGCCGACGGCGUGGGCAAGAGCGCCCUGACCAUCCAGCUGAUCCAG (KRAS G12D 25mer nucleotide sequence) 191AUGACCGAGUACAAGCUGGUGGUGGUGGGCGCCGUGGGCGUGGGCAAGAGCGCCCUGACCAUCCAGCUGAUCCAG (KRAS G12V 25mer nucleotide sequence) 192AUGACCGAGUACAAGCUGGUGGUGGUGGGCGCCGGCGACGUGGGCAAGAGCGCCCUGACCAUCCAGCUGAUCCAG (KRAS G13D 25mer nucleotide sequence) 193AUGACCGAGUACAAGUUAGUGGUUGUGGGCGCCGACGGCGUGGGCAAGAGCGCCCUCACCAUCCAGCUUAUCCAGAUGACGGAAUAUAAGUUAGUAGUAGUGGGAGCCGACGGUGUCGGCAAGUCCGCUUUGACCAUUCAACUUAUUCAGAUGACAGAGUAUAAGCUGGUCGUUGUAGGCGCAGACGGCGUUGGAAAGUCGGCACUGACGAUCCAGUUGAUCCAG (KRAS G12D 25mer^3 nucleotide sequence) 194AUGACCGAGUACAAGCUCGUCGUGGUGGGCGCCGUGGGCGUGGGCAAGAGCGCCCUAACCAUCCAGUUGAUCCAGAUGACCGAAUAUAAGCUCGUGGUAGUCGGAGCGGUGGGCGUUGGCAAGUCAGCGCUAACAAUACAACUAAUCCAAAUGACCGAAUACAAGCUAGUUGUAGUCGGUGCCGUCGGCGUUGGAAAGUCAGCCCUUACAAUUCAGCUCAUUCAG (KRAS G12V 25mer^3 nucleotide sequence) 195AUGACCGAGUACAAGCUCGUAGUGGUUGGCGCCGGCGACGUGGGCAAGAGCGCCCUAACCAUCCAGCUCAUCCAGAUGACAGAAUAUAAGCUUGUGGUUGUGGGAGCAGGAGACGUGGGAAAGAGUGCGUUGACGAUUCAACUCAUACAGAUGACCGAAUACAAGUUGGUGGUGGUCGGCGCAGGUGACGUUGGUAAGUCUGCACUAACUAUACAACUGAUCCAG (KRAS G13D 25mer^3 nucleotide sequence) 196AUGACCGAGUACAAGCUGGUGGUGGUGGGCGCCUGCGGCGUGGGCAAGAGCGCCCUGACCAUCCAGCUGAUCCAG (KRAS G12C 25mer nucleotide sequence) 197AUGACCGAGUACAAGCUCGUGGUUGUUGGCGCCUGCGGCGUGGGCAAGAGCGCCCUCACCAUCCAGCUCAUCCAGAUGACAGAGUAUAAGUUAGUCGUUGUCGGAGCUUGCGGAGUUGGAAAGUCGGCGCUCACCAUUCAACUCAUACAAAUGACAGAAUAUAAGUUAGUGGUGGUGGGUGCGUGUGGCGUUGGCAAGAGUGCGCUUACUAUCCAGCUCAUUCAG (KRAS G12C 25mer^3 nucleotide sequence) 198AUGACCGAGUACAAGCUGGUGGUGGUGGGCGCCGGCGGCGUGGGCAAGAGCGCCCUGACCAUCCAGCUGAUCCAG (KRAS WT 25mer nucleotide sequence) 199GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC (5′ UTR sequence; nopromoter) 200AUGACCGAGUACAAGCUCGUUGUAGUCGGCGCCGACGGCGUGGGCAAGAGCGCCUUGACCAUCCAGUUGAUCCAGAUGACCGAAUAUAAGUUGGUGGUGGUAGGCGCAGUGGGAGUUGGCAAGUCAGCACUCACAAUUCAGCUCAUUCAAAUGACAGAAUACAAGUUAGUCGUUGUAGGAGCAGGCGACGUCGGCAAGAGUGCCUUAACCAUUCAACUAAUCCAG (KRAS(G12D G12V G13D) 75mer “3MUT” nt. seq) 201AUGCCUCACAGCAGCCUGCACCCUAGCAUCCCUUGCCCUAGAGGCCACGGCGCCCAGAAGGCCGCCCUGGUGCUGCUGAGCGCCUGCCUGGUGACCCUGUGGGGCCUGGGCGAGCCUCCUGAGCACACCCUGAGAUACCUGGUGCUGCACCUGGCCAGCCUGCAGCUGGGCCUGCUGCUGAACGGCGUGUGCAGCCUGGCCGAGGAGCUGAGACACAUCCACAGCAGAUACAGAGGCAGCUACUGGAGAACCGUGAGAGCCUGCCUGGGCUGCCCUCUGAGAAGAGGCGCCCUGCUGCUGCUGAGCAUCUACUUCUACUACAGCCUGCCUAACGCCGUGGGCCCUCCUUUCACCUGGAUGCUGGCCCUGCUGGGCCUGAGCCAGGCCCUGAACAUCCUGCUGGGCCUGAAGGGCCUGGCCCCUGCCGAGAUCAGCGCCGUGUGCGAGAAGGGCAACUUCAACGUGGCCCACGGCCUGGCCUGGAGCUACUACAUCGGCUACCUGAGACUGAUCCUGCCUGAGCUGCAGGCCAGAAUCAGAACCUACAACCAGCACUACAACAACCUGCUGAGAGGCGCCGUGAGCCAGAGACUGUACAUCCUGCUGCCUCUGGACUGCGGCGUGCCUGACAACCUGAGCAUGGCCGACCCUAACAUCAGAUUCCUGGACAAGCUGCCUCAGCAGACCGGCGACCACGCCGGCAUCAAGGACAGAGUGUACAGCAACAGCAUCUACGAGCUGCUGGAGAACGGCCAGAGAGCCGGCACCUGCGUGCUGGAGUACGCCACCCCUCUGCAGACCCUGUUCGCCAUGAGCCAGUACAGCCAGGCCGGCUUCAGCAGAGAGGACACCCUGGAGCAGGCCAAGCUGUUCUGCAGAACCCUGGAGGACAUCCUGGCCGACGCCCCUGAGAGCCAGAACAACUGCAGACUGAUCGCCUACCAGGAGCCUGCCGACGACAGCAGCUUCAGCCUGAGCCAGGAGGUGCUGAGACACCUGAGACAGGAGGAGAAGGAGGAGGUGACCGUGGGCAGCCUGAAGACCAGCGCCGUGCCUAGCACCAGCACCAUGAGCCAGGAGCCUGAGCUGCUGAUCAGCGGCAUGGAGAAGCCUCUGCCUCUGAGAACCGACUUCAGC (Hu STING(R284T); no epitope tag; nucleotide sequence) 202AUGCCCCACAGCAGCCUGCACCCCUCCAUCCCCUGUCCCAGAGGCCACGGCGCCCAGAAGGCCGCCCUGGUGCUGCUGAGCGCCUGCCUGGUGACCUUAUGGGGCCUGGGCGAGCCCCCCGAGCACACCCUGAGAUACCUGGUCCUGCACCUGGCCAGCCUCCAGCUGGGCCUGCUGCUCAACGGCGUGUGUAGCCUGGCCGAGGAGCUGAGACACAUCCACAGCAGAUACAGAGGCAGCUACUGGAGAACCGUGAGAGCCUGCCUGGGUUGCCCACUGAGAAGAGGAGCUCUGCUGCUGCUGAGCAUCUACUUCUACUACUCGCUGCCCAACGCUGUGGGCCCCCCCUUCACCUGGAUGCUGGCCCUGCUGGGUCUGAGCCAGGCCCUGAACAUCCUCCUGGGCCUGAAGGGCCUGGCCCCCGCCGAGAUAAGCGCCGUUUGCGAGAAGGGCAACUUCAACGUGGCCCAUGGCCUGGCCUGGAGCUACUACAUCGGCUACUUACGCCUGAUCCUGCCCGAGCUGCAGGCCAGAAUCAGAACCUACAACCAGCAUUACAACAACCUGCUGAGAGGCGCCGUGAGCCAGAGACUGUAUAUCCUGCUGCCCCUGGACUGCGGCGUGCCCGACAACCUGAGCAUGGCCGACCCCAACAUCAGAUUCCUGGACAAGCUCCCCCAGCAGACCGGCGACCACGCCGGAAUCAAAGACAGAGUGUAUAGCAACAGCAUCUACGAGCUGCUGGAGAACGGCCAGAGAGCCGGCACCUGCGUACUGGAGUACGCCACCCCCUUGCAGACCCUGUUUGCCAUGAGCCAGUACAGCCAGGCCGGCUUCAGCAGAGAGGACAUGCUGGAGCAGGCCAAGCUGUUCUGCAGAACCCUGGAGGACAUCCUGGCCGACGCCCCCGAGAGCCAGAACAACUGCAGACUGAUCGCCUACCAAGAGCCCGCCGACGACAGCAGCUUCAGCUUAAGCCAGGAGGUGCUGAGACAUCUGAGACAGGAGGAGAAGGAGGAGGUGACCGUGGGCAGCCUCAAGACCAGCGCUGUGCCCUCUACCAGCACCAUGAGCCAGGAGCCCGAGCUGCUGAUCAGCGGCAUGGAGAAGCCCCUGCCCCUGAGAACAGACUUCAGC (hu STING (R284M); no epitope tag; nucleotide sequence) 203AUGCCCCAUAGCAGCCUGCACCCCAGCAUCCCCUGCCCCAGAGGCCACGGCGCCCAGAAGGCCGCCCUGGUCCUGCUGAGCGCAUGCCUGGUCACCCUGUGGGGCCUGGGCGAGCCCCCCGAGCACACCCUGAGAUACCUGGUGCUGCACCUCGCCAGCCUGCAGCUGGGCCUGCUGCUGAACGGCGUGUGCAGCCUGGCCGAGGAGCUGAGACACAUCCACAGCAGAUAUAGAGGCAGCUACUGGAGAACCGUGAGAGCUUGCCUCGGCUGCCCCCUGAGAAGAGGCGCCCUGCUGCUGCUGAGCAUCUACUUUUACUACAGCCUGCCCAACGCUGUGGGCCCCCCUUUCACGUGGAUGCUCGCCCUGCUGGGACUGAGCCAGGCCCUGAACAUCCUGCUGGGCCUUAAGGGCCUAGCCCCCGCCGAGAUCAGCGCCGUGUGCGAGAAGGGCAACUUCAAUGUGGCCCACGGCCUGGCCUGGAGCUACUACAUCGGCUACCUGAGACUGAUCCUGCCCGAGCUGCAGGCCAGAAUCAGAACCUACAAUCAGCACUACAACAACCUGCUGAGAGGCGCCGUGAGCCAGAGACUGUACAUCCUGCUGCCCCUGGACUGCGGCGUGCCCGACAACCUCAGCAUGGCCGACCCCAACAUCAGAUUCCUGGACAAGCUGCCCCAGCAGACCGGCGACCACGCCGGCAUCAAGGAUCGCGUGUACAGCAACAGCAUCUACGAGCUGCUGGAAAACGGCCAGAGAGCCGGAACCUGCGUGCUGGAGUACGCCACACCCCUGCAGACCCUGUUCGCCAUGAGCCAGUACAGCCAGGCCGGCUUCAGCAGAGAGGACAAGCUGGAGCAGGCCAAGCUGUUCUGCAGAACCCUGGAGGAUAUCCUCGCCGACGCCCCCGAGAGCCAGAACAACUGCAGGCUGAUCGCGUACCAGGAGCCCGCUGACGACAGCAGCUUUAGCCUGAGCCAGGAGGUGCUGAGACAUCUGCGUCAAGAGGAAAAGGAGGAGGUGACCGUGGGCUCCCUGAAGACCAGCGCCGUGCCCAGCACCAGCACCAUGAGCCAGGAGCCCGAGCUGCUGAUCAGCGGCAUGGAGAAGCCACUGCCCCUCAGAACCGACUUCAGC (Hu STING (R284K); no epitope tag; nucleotide sequence) 204AUGCCUCACAGCAGCCUGCACCCUAGCAUCCCUUGCCCUAGAGGCCACGGCGCCCAGAAGGCCGCCCUGGUGCUGCUGAGCGCCUGCCUGGUGACCCUGUGGGGCCUGGGCGAGCCUCCUGAGCACACCCUGAGAUACCUGGUGCUGCACCUGGCCAGCCUGCAGCUGGGCCUGCUGCUGAACGGCGUGUGCAGCCUGGCCGAGGAGCUGAGACACAUCCACAGCAGAUACAGAGGCAGCUACUGGAGAACCGUGAGAGCCUGCCUGGGCUGCCCUCUGAGAAGAGGCGCCCUGCUGCUGCUGAGCAUCUACUUCUACUACAGCCUGCCUAACGCCGUGGGCCCUCCUUUCACCUGGAUGCUGGCCCUGCUGGGCCUGAGCCAGGCCCUGAACAUCCUGCUGGGCCUGAAGGGCCUGGCCCCUGCCGAGAUCAGCGCCGUGUGCGAGAAGGGCAACUUCAGCGUGGCCCACGGCCUGGCCUGGAGCUACUACAUCGGCUACCUGAGACUGAUCCUGCCUGAGCUGCAGGCCAGAAUCAGAACCUACAACCAGCACUACAACAACCUGCUGAGAGGCGCCGUGAGCCAGAGACUGUACAUCCUGCUGCCUCUGGACUGCGGCGUGCCUGACAACCUGAGCAUGGCCGACCCUAACAUCAGAUUCCUGGACAAGCUGCCUCAGCAGACCGGCGACCACGCCGGCAUCAAGGACAGAGUGUACAGCAACAGCAUCUACGAGCUGCUGGAGAACGGCCAGAGAGCCGGCACCUGCGUGCUGGAGUACGCCACCCCUCUGCAGACCCUGUUCGCCAUGAGCCAGUACAGCCAGGCCGGCUUCAGCAGAGAGGACAGACUGGAGCAGGCCAAGCUGUUCUGCAGAACCCUGGAGGACAUCCUGGCCGACGCCCCUGAGAGCCAGAACAACUGCAGACUGAUCGCCUACCAGGAGCCUGCCGACGACAGCAGCUUCAGCCUGAGCCAGGAGGUGCUGAGACACCUGAGACAGGAGGAGAAGGAGGAGGUGACCGUGGGCAGCCUGAAGACCAGCGCCGUGCCUAGCACCAGCACCAUGAGCCAGGAGCCUGAGCUGCUGAUCAGCGGCAUGGAGAAGCCUCUGCCUCUGAGAACCGACUUCAGC (Hu STING(N154S); no epitope tag; nucleotide sequence) 205AUGCCUCACAGCAGCCUGCACCCUAGCAUCCCUUGCCCUAGAGGCCACGGCGCCCAGAAGGCCGCCCUGGUGCUGCUGAGCGCCUGCCUGGUGACCCUGUGGGGCCUGGGCGAGCCUCCUGAGCACACCCUGAGAUACCUGGUGCUGCACCUGGCCAGCCUGCAGCUGGGCCUGCUGCUGAACGGCGUGUGCAGCCUGGCCGAGGAGCUGAGACACAUCCACAGCAGAUACAGAGGCAGCUACUGGAGAACCGUGAGAGCCUGCCUGGGCUGCCCUCUGAGAAGAGGCGCCCUGCUGCUGCUGAGCAUCUACUUCUACUACAGCCUGCCUAACGCCGUGGGCCCUCCUUUCACCUGGAUGCUGGCCCUGCUGGGCCUGAGCCAGGCCCUGAACAUCCUGCUGGGCCUGAAGGGCCUGGCCCCUGCCGAGAUCAGCGCCCUGUGCGAGAAGGGCAACUUCAACGUGGCCCACGGCCUGGCCUGGAGCUACUACAUCGGCUACCUGAGACUGAUCCUGCCUGAGCUGCAGGCCAGAAUCAGAACCUACAACCAGCACUACAACAACCUGCUGAGAGGCGCCGUGAGCCAGAGACUGUACAUCCUGCUGCCUCUGGACUGCGGCGUGCCUGACAACCUGAGCAUGGCCGACCCUAACAUCAGAUUCCUGGACAAGCUGCCUCAGCAGACCGGCGACCACGCCGGCAUCAAGGACAGAGUGUACAGCAACAGCAUCUACGAGCUGCUGGAGAACGGCCAGAGAGCCGGCACCUGCGUGCUGGAGUACGCCACCCCUCUGCAGACCCUGUUCGCCAUGAGCCAGUACAGCCAGGCCGGCUUCAGCAGAGAGGACAGACUGGAGCAGGCCAAGCUGUUCUGCAGAACCCUGGAGGACAUCCUGGCCGACGCCCCUGAGAGCCAGAACAACUGCAGACUGAUCGCCUACCAGGAGCCUGCCGACGACAGCAGCUUCAGCCUGAGCCAGGAGGUGCUGAGACACCUGAGACAGGAGGAGAAGGAGGAGGUGACCGUGGGCAGCCUGAAGACCAGCGCCGUGCCUAGCACCAGCACCAUGAGCCAGGAGCCUGAGCUGCUGAUCAGCGGCAUGGAGAAGCCUCUGCCUCUGAGAACCGACUUCAGC (Hu STING(V147L); no epitope tag; nucleotide sequence) 206AUGCCUCACAGCAGCCUGCACCCUAGCAUCCCUUGCCCUAGAGGCCACGGCGCCCAGAAGGCCGCCCUGGUGCUGCUGAGCGCCUGCCUGGUGACCCUGUGGGGCCUGGGCGAGCCUCCUGAGCACACCCUGAGAUACCUGGUGCUGCACCUGGCCAGCCUGCAGCUGGGCCUGCUGCUGAACGGCGUGUGCAGCCUGGCCGAGGAGCUGAGACACAUCCACAGCAGAUACAGAGGCAGCUACUGGAGAACCGUGAGAGCCUGCCUGGGCUGCCCUCUGAGAAGAGGCGCCCUGCUGCUGCUGAGCAUCUACUUCUACUACAGCCUGCCUAACGCCGUGGGCCCUCCUUUCACCUGGAUGCUGGCCCUGCUGGGCCUGAGCCAGGCCCUGAACAUCCUGCUGGGCCUGAAGGGCCUGGCCCCUGCCGAGAUCAGCGCCGUGUGCGAGAAGGGCAACUUCAACGUGGCCCACGGCCUGGCCUGGAGCUACUACAUCGGCUACCUGAGACUGAUCCUGCCUGAGCUGCAGGCCAGAAUCAGAACCUACAACCAGCACUACAACAACCUGCUGAGAGGCGCCGUGAGCCAGAGACUGUACAUCCUGCUGCCUCUGGACUGCGGCGUGCCUGACAACCUGAGCAUGGCCGACCCUAACAUCAGAUUCCUGGACAAGCUGCCUCAGCAGACCGGCGACCACGCCGGCAUCAAGGACAGAGUGUACAGCAACAGCAUCUACGAGCUGCUGGAGAACGGCCAGAGAGCCGGCACCUGCGUGCUGGAGUACGCCACCCCUCUGCAGACCCUGUUCGCCAUGAGCCAGUACAGCCAGGCCGGCUUCAGCAGAGAGGACAGACUGGAGCAGGCCAAGCUGUUCUGCAGAACCCUGGAGGACAUCCUGGCCGACGCCCCUGAGAGCCAGAACAACUGCAGACUGAUCGCCUACCAGCAGCCUGCCGACGACAGCAGCUUCAGCCUGAGCCAGGAGGUGCUGAGACACCUGAGACAGGAGGAGAAGGAGGAGGUGACCGUGGGCAGCCUGAAGACCAGCGCCGUGCCUAGCACCAGCACCAUGAGCCAGGAGCCUGAGCUGCUGAUCAGCGGCAUGGAGAAGCCUCUGCCUCUGAGAACCGACUUCAGC (Hu STING (E315Q); no epitope tag; nucleotide sequence) 207AUGCCUCACAGCAGCCUGCACCCUAGCAUCCCUUGCCCUAGAGGCCACGGCGCCCAGAAGGCCGCCCUGGUGCUGCUGAGCGCCUGCCUGGUGACCCUGUGGGGCCUGGGCGAGCCUCCUGAGCACACCCUGAGAUACCUGGUGCUGCACCUGGCCAGCCUGCAGCUGGGCCUGCUGCUGAACGGCGUGUGCAGCCUGGCCGAGGAGCUGAGACACAUCCACAGCAGAUACAGAGGCAGCUACUGGAGAACCGUGAGAGCCUGCCUGGGCUGCCCUCUGAGAAGAGGCGCCCUGCUGCUGCUGAGCAUCUACUUCUACUACAGCCUGCCUAACGCCGUGGGCCCUCCUUUCACCUGGAUGCUGGCCCUGCUGGGCCUGAGCCAGGCCCUGAACAUCCUGCUGGGCCUGAAGGGCCUGGCCCCUGCCGAGAUCAGCGCCGUGUGCGAGAAGGGCAACUUCAACGUGGCCCACGGCCUGGCCUGGAGCUACUACAUCGGCUACCUGAGACUGAUCCUGCCUGAGCUGCAGGCCAGAAUCAGAACCUACAACCAGCACUACAACAACCUGCUGAGAGGCGCCGUGAGCCAGAGACUGUACAUCCUGCUGCCUCUGGACUGCGGCGUGCCUGACAACCUGAGCAUGGCCGACCCUAACAUCAGAUUCCUGGACAAGCUGCCUCAGCAGACCGGCGACCACGCCGGCAUCAAGGACAGAGUGUACAGCAACAGCAUCUACGAGCUGCUGGAGAACGGCCAGAGAGCCGGCACCUGCGUGCUGGAGUACGCCACCCCUCUGCAGACCCUGUUCGCCAUGAGCCAGUACAGCCAGGCCGGCUUCAGCAGAGAGGACAGACUGGAGCAGGCCAAGCUGUUCUGCAGAACCCUGGAGGACAUCCUGGCCGACGCCCCUGAGAGCCAGAACAACUGCAGACUGAUCGCCUACCAGGAGCCUGCCGACGACAGCAGCUUCAGCCUGAGCCAGGAGGUGCUGAGACACCUGAGACAGGAGGAGAAGGAGGAGGUGACCGUGGGCAGCCUGAAGACCAGCGCCGUGCCUAGCACCAGCACCAUGAGCCAGGAGCCUGAGCUGCUGAUCAGCGGCAUGGAGAAGCCUCUGCCUCUGGCCACCGACUUCAGC (Hu STING (R375A); no epitope tag; nucleotide sequence) 208AUGCCUCACAGCAGCCUGCACCCUAGCAUCCCUUGCCCUAGAGGCCACGGCGCCCAGAAGGCCGCCCUGGUGCUGCUGAGCGCCUGCCUGGUGACCCUGUGGGGCCUGGGCGAGCCUCCUGAGCACACCCUGAGAUACCUGGUGCUGCACCUGGCCAGCCUGCAGCUGGGCCUGCUGCUGAACGGCGUGUGCAGCCUGGCCGAGGAGCUGAGACACAUCCACAGCAGAUACAGAGGCAGCUACUGGAGAACCGUGAGAGCCUGCCUGGGCUGCCCUCUGAGAAGAGGCGCCCUGCUGCUGCUGAGCAUCUACUUCUACUACAGCCUGCCUAACGCCGUGGGCCCUCCUUUCACCUGGAUGCUGGCCCUGCUGGGCCUGAGCCAGGCCCUGAACAUCCUGCUGGGCCUGAAGGGCCUGGCCCCUGCCGAGAUCAGCGCCCUGUGCGAGAAGGGCAACUUCAGCAUGGCCCACGGCCUGGCCUGGAGCUACUACAUCGGCUACCUGAGACUGAUCCUGCCUGAGCUGCAGGCCAGAAUCAGAACCUACAACCAGCACUACAACAACCUGCUGAGAGGCGCCGUGAGCCAGAGACUGUACAUCCUGCUGCCUCUGGACUGCGGCGUGCCUGACAACCUGAGCAUGGCCGACCCUAACAUCAGAUUCCUGGACAAGCUGCCUCAGCAGACCGGCGACCACGCCGGCAUCAAGGACAGAGUGUACAGCAACAGCAUCUACGAGCUGCUGGAGAACGGCCAGAGAGCCGGCACCUGCGUGCUGGAGUACGCCACCCCUCUGCAGACCCUGUUCGCCAUGAGCCAGUACAGCCAGGCCGGCUUCAGCAGAGAGGACAGACUGGAGCAGGCCAAGCUGUUCUGCAGAACCCUGGAGGACAUCCUGGCCGACGCCCCUGAGAGCCAGAACAACUGCAGACUGAUCGCCUACCAGGAGCCUGCCGACGACAGCAGCUUCAGCCUGAGCCAGGAGGUGCUGAGACACCUGAGACAGGAGGAGAAGGAGGAGGUGACCGUGGGCAGCCUGAAGACCAGCGCCGUGCCUAGCACCAGCACCAUGAGCCAGGAGCCUGAGCUGCUGAUCAGCGGCAUGGAGAAGCCUCUGCCUCUGAGAACCGACUUCAGC (Hu STING(V147L/N154S/V155M); no epitope tag; nucleotidesequence) 209AUGCCUCACAGCAGCCUGCACCCUAGCAUCCCUUGCCCUAGAGGCCACGGCGCCCAGAAGGCCGCCCUGGUGCUGCUGAGCGCCUGCCUGGUGACCCUGUGGGGCCUGGGCGAGCCUCCUGAGCACACCCUGAGAUACCUGGUGCUGCACCUGGCCAGCCUGCAGCUGGGCCUGCUGCUGAACGGCGUGUGCAGCCUGGCCGAGGAGCUGAGACACAUCCACAGCAGAUACAGAGGCAGCUACUGGAGAACCGUGAGAGCCUGCCUGGGCUGCCCUCUGAGAAGAGGCGCCCUGCUGCUGCUGAGCAUCUACUUCUACUACAGCCUGCCUAACGCCGUGGGCCCUCCUUUCACCUGGAUGCUGGCCCUGCUGGGCCUGAGCCAGGCCCUGAACAUCCUGCUGGGCCUGAAGGGCCUGGCCCCUGCCGAGAUCAGCGCCCUGUGCGAGAAGGGCAACUUCAGCAUGGCCCACGGCCUGGCCUGGAGCUACUACAUCGGCUACCUGAGACUGAUCCUGCCUGAGCUGCAGGCCAGAAUCAGAACCUACAACCAGCACUACAACAACCUGCUGAGAGGCGCCGUGAGCCAGAGACUGUACAUCCUGCUGCCUCUGGACUGCGGCGUGCCUGACAACCUGAGCAUGGCCGACCCUAACAUCAGAUUCCUGGACAAGCUGCCUCAGCAGACCGGCGACCACGCCGGCAUCAAGGACAGAGUGUACAGCAACAGCAUCUACGAGCUGCUGGAGAACGGCCAGAGAGCCGGCACCUGCGUGCUGGAGUACGCCACCCCUCUGCAGACCCUGUUCGCCAUGAGCCAGUACAGCCAGGCCGGCUUCAGCAGAGAGGACAUGCUGGAGCAGGCCAAGCUGUUCUGCAGAACCCUGGAGGACAUCCUGGCCGACGCCCCUGAGAGCCAGAACAACUGCAGACUGAUCGCCUACCAGGAGCCUGCCGACGACAGCAGCUUCAGCCUGAGCCAGGAGGUGCUGAGACACCUGAGACAGGAGGAGAAGGAGGAGGUGACCGUGGGCAGCCUGAAGACCAGCGCCGUGCCUAGCACCAGCACCAUGAGCCAGGAGCCUGAGCUGCUGAUCAGCGGCAUGGAGAAGCCUCUGCCUCUGAGAACCGACUUCAGC (Hu STING(R284M/V147L/N154S/V155M); no epitope tag; nucleotidesequence) 210UGAUAAUAGGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCCAAACACCAUUGUCACACUCCAGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC(3′ UTR used in STING V155M construct, containing miR122 binding site)211AUGGAGACCCCCAAGCCUAGAAUCCUGCCCUGGCUGGUGAGCCAGCUGGACCUGGGCCAGCUGGAGGGCGUAGCCUGGCUGGACGAGAGCAGAACCAGAUUCAGAAUCCCCUGGAAGCACGGCCUGAGACAAGACGCCCAGAUGGCCGACUUCGGCAUCUUCCAGGCCUGGGCCGAGGCCAGCGGCGCCUACACCCCUGGCAAGGAUAAGCCCGAUGUGAGCACCUGGAAGAGAAACUUCAGAAGCGCCCUGAACAGAAAGGAGGUGCUGAGACUGGCCGCCGACAAUAGCAAGGACCCCUACGACCCCCACAAGGUGUACGAGUUCGUUACCCCCGGCGCCAGGGACUUCGUGCACCUGGGCGCCAGCCCCGACACCAACGGCAAGAGCAGCCUGCCCCACAGCCAGGAGAACCUGCCCAAGCUGUUCGAUGGCCUGAUCCUGGGCCCCCUGAAGGACGAGGGCAGCAGCGACCUGGCCAUCGUGAGCGACCCUAGCCAGCAGCUGCCCUCCCCCAACGUGAACAACUUCCUGAACCCCGCCCCCCAGGAGAACCCCCUGAAGCAACUGCUGGCCGAGGAGCAGUGGGAGUUCGAGGUGACCGCCUUCUACAGAGGCAGACAGGUGUUCCAGCAGACCCUGUUCUGCCCCGGCGGCCUGAGACUGGUAGGCAGCACCGCUGACAUGACCCUGCCCUGGCAGCCCGUGACCCUGCCCGACCCCGAAGGCUUUCUGACCGACAAGCUGGUGAAGGAGUACGUCGGCCAAGUGCUGAAGGGCCUGGGCAACGGCCUGGCCCUGUGGCAGGCCGGCCAGUGCCUGUGGGCCCAGAGACUCGGCCACAGCCACGCCUUCUGGGCCCUGGGCGAGGAACUCCUGCCCGAUAGCGGCAGAGGCCCCGACGGCGAGGUGCACAAGGACAAGGACGGCGCCGUGUUCGACCUGCGCCCCUUCGUGGCCGACCUGAUCGCCUUCAUGGAGGGCAGCGGCCACAGCCCCAGAUAUACCCUGUGGUUCUGCAUGGGCGAGAUGUGGCCCCAGGACCAGCCCUGGGUGAAGAGACUGGUGAUGGUGAAGGUGGUGCCCACCUGCCUGAAAGAGCUGCUGGAGAUGGCCAGAGAGGGCGGCGCCAGCUCCCUGAAAACCGUGGACCUGCACAUUGACAACAGCCAGCCCAUCAGCCUGACCAGCGACCAGUACAAGGCCUACCUGCAGGACCUGGUGGAGGACAUGGACUUCCAGGCCACCGGCAACAUC (super mouseIRF3 S396D; no epitope tag) 212AUGGGCACCCCCAAGCCCAGAAUCCUGCCCUGGCUGGUGAGCCAGCUGGACCUGGGCCAGCUGGAGGGAGUGGCCUGGGUGAACAAGAGCAGAACCAGAUUCAGAAUCCCCUGGAAGCACGGCCUCAGACAGGACGCCCAGCAGGAGGACUUCGGCAUUUUUCAGGCUUGGGCCGAGGCCACCGGCGCCUACGUGCCCGGCAGAGACAAGCCCGACCUGCCCACCUGGAAAAGAAACUUCAGAAGCGCCUUGAAUAGAAAGGAGGGCCUGAGACUGGCCGAGGACAGAAGCAAGGACCCCCACGACCCUCACAAGAUCUACGAGUUCGUGAAUAGCGGCGUGGGCGACUUUAGCCAGCCCGACACCAGCCCCGACACCAACGGCGGCGGCAGCACCAGCGACACGCAGGAGGACAUCCUGGAUGAACUGCUGGGCAACAUGGUGCUGGCCCCCCUGCCCGAUCCCGGCCCCCCUUCGCUUGCCGUGGCCCCCGAGCCCUGCCCCCAGCCCCUGAGAAGCCCCUCUCUGGAUAACCCCACCCCCUUCCCCAACCUGGGCCCCAGCGAGAAUCCACUGAAGAGACUUCUGGUCCCCGGCGAGGAGUGGGAGUUCGAGGUGACCGCCUUCUACAGAGGCAGACAGGUGUUCCAGCAGACCAUCAGCUGCCCCGAAGGCCUGAGAUUAGUGGGCAGCGAAGUGGGCGACAGGACCCUGCCCGGGUGGCCCGUGACCCUGCCCGAUCCCGGCAUGAGCCUGACCGACAGAGGUGUGAUGAGCUACGUGAGACACGUGCUGAGCUGCCUGGGCGGCGGCCUGGCACUGUGGAGAGCCGGCCAGUGGCUGUGGGCCCAGAGACUGGGCCACUGCCACACCUACUGGGCCGUGAGCGAGGAGCUGCUGCCCAACAGCGGCCACGGCCCCGACGGCGAGGUGCCCAAGGACAAGGAAGGGGGCGUGUUCGACCUGGGCCCCUUCAUCGUAGACCUGAUCACCUUUACCGAGGGCAGCGGCAGGAGCCCCAGAUACGCCCUGUGGUUCUGCGUGGGCGAAAGCUGGCCCCAGGACCAGCCCUGGACCAAGAGACUGGUGAUGGUGAAGGUAGUGCCCACCUGCCUGAGAGCCUUAGUGGAGAUGGCCAGAGUGGGCGGGGCCAGCAGCCUGGAGAACACCGUGGAUCUUCACAUCGACAACAGCCACCCCCUGAGCCUGACCAGCGACCAGUACAAGGCCUACCUGCAGGACCUGGUGGAGGGCAUGGACUUCCAGGGCCCCGGCGAGACC (super human IRF3 S396D; no epitope tag) 213AUGGCGCUGGCCCCCGAAAGAGCCGCCCCCAGAGUCCUCUUCGGCGAAUGGCUCCUUGGCGAAAUUUCGUCGGGCUGCUACGAGGGCUUACAAUGGCUGGAUGAGGCGAGAACCUGUUUCAGGGUGCCCUGGAAACACUUCGCCAGAAAGGAUCUAAGCGAAGCAGAUGCUAGAAUUUUUAAGGCUUGGGCCGUGGCCAGGGGAAGAUGGCCCCCCUCGAGCAGAGGCGGCGGCCCUCCCCCCGAGGCAGAAACGGCCGAGAGAGCCGGAUGGAAAACCAAUUUCAGAUGCGCCCUGAGAUCUACAAGAAGAUUCGUGAUGCUUAGAGACAACAGCGGAGAUCCCGCCGAUCCCCAUAAGGUGUAUGCCCUGUCCCGGGAGCUGUGCUGGAGGGAAGGGCCUGGCACUGACCAGACCGAAGCCGAAGCCCCCGCGGCCGUGCCGCCGCCCCAAGGAGGCCCACCAGGCCCUUUCCUCGCUCACACCCACGCCGGUCUGCAAGCCCCGGGACCUCUACCUGCCCCUGCCGGCGAUAAAGGCGACCUGUUGCUGCAGGCCGUCCAACAGAGCUGCCUGGCCGAUCAUCUGCUCACAGCCAGCUGGGGCGCUGACCCCGUCCCAACAAAGGCCCCCGGUGAGGGCCAAGAAGGCCUGCCUCUGACCGGCGCCUGUGCCGGCGGCCCUGGCCUGCCUGCUGGCGAGCUGUACGGAUGGGCUGUCGAAACCACUCCCUCCCCCGGCCCCCAACCUGCGGCCCUGACAACCGGCGAGGCAGCCGCACCCGAAAGCCCCCACCAGGCCGAACCCUACCUCAGUCCCAGCCCCUCCGCCUGCACCGCUGUGCAGGAGCCCAGCCCCGGUGCUCUGGACGUAACAAUCAUGUACAAAGGCAGAACCGUGCUUCAGAAGGUGGUUGGACACCCCUCCUGUACUUUUCUCUACGGCCCCCCCGACCCUGCCGUGAGAGCUACCGACCCGCAACAGGUGGCCUUUCCCUCGCCCGCCGAACUGCCCGAUCAAAAACAGCUGAGAUACACCGAGGAGCUGCUGAGACACGUGGCGCCGGGCUUACACCUAGAGUUGAGAGGCCCCCAACUCUGGGCCAGACGCAUGGGCAAGUGUAAGGUGUACUGGGAGGUCGGGGGCCCUCCCGGCUCUGCCAGCCCCAGCACCCCUGCUUGUCUCUUGCCCAGAAACUGUGAUACCCCCAUCUUCGACUUCCGUGUAUUUUUCCAGGAACUGGUCGAGUUUAGAGCCAGACAGAGACGAGGCAGCCCCAGAUAUACAAUCUACCUCGGCUUCGGCCAGGACCUGAGUGCCGGCAGACCUAAGGAGAAGUCGCUGGUCCUAGUGAAGUUAGAGCCCUGGCUAUGUAGAGUGCACCUGGAGGGCACCCAGAGAGAAGGAGUGAGCAGCCUGGACAGCAGCAGCCUGAGUCUGUGCCUGAGCUCCGCCAACUCGCUGUAUGAUGACAUCGAGUGUUUCCUCAUGGAGCUGGAGCAGCCCGCC (Wild-type Hu IRF7 isoform A; P037 withoutepitope tag) 214AUGGCCCUUGCCCCUGAGCGGGCCGCCCCCAGAGUGUUAUUCGGCGAGUGGCUGCUGGGCGAGAUCAGCAGCGGCUGCUACGAGGGACUGCAGUGGCUGGACGAGGCUAGAACCUGCUUCAGAGUGCCCUGGAAGCAUUUCGCCAGAAAAGACCUGAGCGAGGCUGAUGCUAGAAUCUUCAAAGCCUGGGCUGUGGCCCGAGGAAGAUGGCCCCCCAGCAGCAGAGGAGGCGGCCCUCCUCCCGAGGCCGAAACCGCAGAGCGUGCUGGCUGGAAAACCAACUUUAGGUGUGCCCUGAGGAGCACCAGAAGAUUCGUUAUGCUCAGAGACAACAGCGGGGACCCCGCCGACCCGCACAAGGUGUACGCCUUAAGUAGGGAGCUGUGCUGGAGAGAGGGACCGGGGACCGACCAAACCGAGGCUGAGGCGCCCGCCGCCGUUCCACCUCCCCAGGGUGGUCCCCCAGGGCCCUUUCUGGCACACACCCACGCCGGAUUACAGGCGCCAGGGCCCUUACCCGCCCCCGCCGGAGACAAAGGCGACCUCCUGCUGCAAGCCGUGCAACAAAGCUGCCUGGCCGAUCACUUACUAACCGCUAGCUGGGGCGCCGAUCCUGUUCCCACCAAGGCCCCCGGUGAAGGGCAAGAAGGACUGCCCUUAACCGGCGCCUGUGCCGGAGGCCCUGGUCUGCCAGCCGGCGAGCUGUACGGUUGGGCUGUCGAAACAACACCCAGUCCGGGCCCACAGCCUGCCGCUCUGACCACCGGCGAAGCCGCCGCCCCCGAGAGCCCACACCAGGCUGAACCCUACCUGAGCCCCAGCCCCAGCGCCUGCACCGCUGUGCAGGAGCCUAGCCCCGGCGCUCUUGAUGUGACAAUAAUGUACAAGGGCAGGACCGUGCUGCAAAAGGUCGUGGGCCAUCCGUCGUGUACCUUUCUGUACGGCCCUCCAGACCCCGCGGUUAGAGCCACCGACCCCCAGCAAGUCGCCUUCCCCUCCCCCGCCGAACUGCCCGACCAAAAGCAGCUGCGGUACACAGAAGAACUACUUAGACACGUGGCCCCCGGUCUGCACUUGGAGCUGAGAGGCCCCCAGCUCUGGGCCAGAAGAAUGGGCAAGUGCAAAGUGUACUGGGAGGUGGGCGGCCCACCCGGCUCAGCUUCGCCCUCCACACCCGCAUGCCUGCUGCCCAGAAAUUGCGACACGCCCAUCUUCGAUUUUAGAGUGUUCUUUCAGGAGUUGGUGGAGUUCAGAGCCAGACAAAGACGCGGCAGCCCCAGAUACACCAUUUACCUCGGCUUCGGCCAGGACCUCAGCGCUGGCAGACCCAAGGAGAAGAGUCUGGUCCUCGUGAAGCUGGAGCCCUGGCUGUGCAGAGUGCACCUGGAGGGCACCCAGCGUGAAGGCGUGAGCAGCCUGGAUUCAAGCGACCUGGACCUAUGCCUAAGCAGCGCUAACUCACUGUACGACGAUAUCGAAUGCUUCCUGAUGGAACUGGAGCAGCCUGCC (constitutively active Hu IRF7 S477D/S479D;P033 without epitope tag) 215AUGGCCCUGGCACCCGAGAGGGCCGCCCCCAGGGUGCUCUUCGGCGAGUGGUUACUAGGCGAAAUUAGCAGCGGCUGCUAUGAAGGCCUUCAGUGGCUGGACGAGGCCAGAACCUGCUUUAGAGUUCCCUGGAAGCACUUCGCCCGGAAAGAUCUCUCUGAAGCCGACGCCAGAAUAUUCAAGGCCUGGGCUGUCGCCAGGGGCAGGUGGCCACCCUCCAGCCGAGGUGGCGGCCCUCCCCCUGAGGCUGAGACUGCGGAAAGGGCGGGCUGGAAGACCAAUUUCAGAUGCGCUCUGAGAAGCACCAGACGUUUUGUGAUGCUAAGAGACAAUAGCGGCGAUCCCGCCGACCCCCAUAAGGUAUACGCACUGAGCCGAGAGCUCUGUUGGAGAGAAGGCCCCGGCACCGACCAGACCGAGGCUGAAGCCCCUGCAGCCGUGCCCCCCCCUCAAGGCGGGCCCCCCGGCCCCUUCCUGGCCCAUACCCAUGCAGGGUUACAAGCACCCGGGCCCUUGCCCGCCCCAGCGGGAGACAAGGGCGACCUCUUACUGCAGGCCGUGCAACAAAGUUGUCUGGCGGACCACCUGCUGACCGCAUCAUGGGGCGCGGAUCCUGUGCCCACCAAGGCACCCGGCGAAGGCCAGGAGGGCCUGCCCUUGACCGGCGCCUGCGCUGGCGGACCCGGCCUACCUGCUGGCGAACUGUAUGGCUGGGCCGUAGAGACGACUCCCAGCCCUGGCCCACAACCCGCGGCUUUGACCACCGGCGAAGCCGCCGCCCCCGAGUCUCCGCACCAGGCCGAGCCUUACCUCAGCCCAAGCCCUAGCGCCUGCACCGCCGUGCAAGAACCUAGCCCCGGAGCCCUGGAUGUGACAAUCAUGUACAAGGGUAGAACCGUACUGCAAAAGGUGGUGGGUCAUCCCAGCUGCACCUUUCUUUACGGCCCACCCGACCCUGCCGUGCGAGCCACAGACCCACAACAGGUCGCCUUCCCAAGCCCCGCCGAACUGCCCGAUCAGAAACAGCUGAGAUAUACAGAGGAGCUUCUGCGGCACGUAGCUCCCGGCCUACAUCUCGAGCUGAGGGGCCCACAACUGUGGGCCAGACGCAUGGGCAAAUGCAAGGUCUACUGGGAAGUGGGAGGCCCCCCCGGCAGCGCAUCUCCCAGCACGCCCGCGUGCCUGCUGCCUAGAAAUUGCGACACCCCCAUCUUUGACUUCCGGGUAUUCUUUCAGGAGCUGGUAGAGUUCAGAGCCAGGCAGCGGAGGGGCUCCCCCAGAUACACAAUCUACCUGGGCUUCGGACAGGACCUGUCCGCCGGCCGCCCCAAGGAAAAGAGCCUGGUGCUGGUGAAGCUGGAGCCCUGGCUGUGUAGGGUACACCUCGAAGGCACCCAGAGAGAAGGAGUGAGCUCGCUUGAUGACAGCGAUCUGUCGGAUUGCCUUAGCAGCGCCAACAGCCUGUAUGAUGAUAUCGAGUGCUUCCUUAUGGAACUGGAGCAGCCCGCC (constitutively active Hu IRF7S475D/S477D/L480D; P034 without epitope tag) 216AUGGCCCUAGCCCCCGAAAGAGCAGCUCCCAGAGUGCUGUUCGGCGAAUGGCUGCUUGGCGAGAUCAGCAGCGGCUGCUACGAAGGCCUGCAGUGGCUGGACGAAGCCCGCACCUGUUUCAGAGUGCCCUGGAAGCACUUCGCUAGAAAGGAUUUGAGCGAGGCUGAUGCUAGAAUCUUUAAGGCUUGGGCUGUGGCAAGAGGCAGAUGGCCGCCUAGUAGCAGAGGGGGCGGACCUCCCCCCGAGGCUGAGACCGCUGAGAGAGCAGGGUGGAAAACCAACUUCAGAUGCGCGCUGAGAAGCACCCGAAGAUUCGUGAUGCUACGUGACAAUAGCGGCGACCCCGCCGACCCCCACAAAGUGUACGCCCUGUCCCGAGAACUUUGCUGGAGAGAGGGACCCGGCACCGAUCAAACAGAGGCUGAGGCCCCGGCCGCUGUACCCCCGCCCCAAGGAGGCCCCCCAGGCCCCUUUCUGGCUCAUACACAUGCCGGCCUGCAGGCACCCGGGCCCCUCCCGGCUCCUGCCGGCGACAAGGGCGAUCUCCUUCUCCAGGCCGUGCAGCAGAGCUGCCUGGCCGAUCACCUGCUGACCGCCUCGUGGGGCGCCGACCCCGUGCCCACCAAAGCCCCGGGUGAAGGCCAAGAGGGGCUCCCUUUAACCGGAGCAUGCGCCGGAGGCCCCGGCCUGCCAGCCGGCGAGUUAUAUGGCUGGGCUGUGGAGACCACACCCUCCCCCGGCCCUCAACCCGCUGCCCUGACCACCGGUGAGGCCGCCGCCCCCGAGAGCCCACACCAGGCCGAACCCUACCUGAGCCCUAGCCCUAGCGCCUGCACCGCCGUGCAAGAACCCAGCCCCGGAGCCCUGGAUGUGACCAUUAUGUACAAGGGCCGGACAGUGCUGCAAAAGGUUGUGGGACACCCGAGCUGCACCUUUCUGUACGGUCCGCCUGACCCCGCCGUGAGAGCCACGGACCCGCAGCAGGUGGCCUUCCCCUCACCCGCGGAGCUGCCCGACCAAAAGCAACUCAGAUACACAGAAGAACUAUUGCGUCACGUCGCGCCCGGCCUGCAUCUGGAGCUGAGAGGCCCCCAGCUCUGGGCCAGAAGGAUGGGCAAAUGCAAGGUGUACUGGGAGGUGGGAGGCCCCCCCGGCAGCGCCAGCCCCAGCACUCCCGCGUGCCUGCUGCCCAGAAAUUGCGACACUCCCAUCUUCGAUUUCAGGGUGUUCUUCCAGGAGCUGGUGGAGUUCAGAGCCAGGCAGAGAAGGGGUAGCCCCAGAUACACAAUCUAUCUAGGCUUUGGACAAGAUCUGAGCGCCGGCCGGCCUAAGGAAAAAAGCCUGGUGCUGGUAAAGCUGGAGCCGUGGCUUUGUAGAGUGCACCUGGAGGGGACGCAGCGAGAGGGCGUGAGCAGCUUAGACGACGAUGACUUGGAUCUGUGUCUCGACAGCGCCAACGACUUGUACGACGACAUCGAGUGCUUCCUGAUGGAACUGGAGCAGCCCGCC (constitutively active Hu IRF7S475D/S476D/S477D/S479D/S483D/S487D; P035 without epitope tag) 217AUGGCCCUGGCCCCCGAGAGAGCCGCCCCCAGAGUGCUCUUCGGCGAGUGGCUGCUGGGCGAGAUAAGCAGCGGCUGCUACGAAGGUCUGCAGUGGCUAGACGAGGCCAGAACCUGCUUUAGAGUGCCCUGGAAGCACUUCGCUCGAAAGGACCUGUCCGAGGCCGAUGCUAGAAUUUUUAAGGCUUGGGCCGUCGCUAGGGGAAGAUGGCCCCCUAGCAGUAGAGGCGGCGGCCCCCCUCCCGAAGCCGAGACGGCCGAGAGGGCCGGCUGGAAAACCAAUUUCAGAUGCGCCCUGAGGAGCACCCGCAGGUUCGUAAUGCUGCGAGACAAUAGCGGCGAUCCUGCGGAUCCUCACAAGGUUUACGCCUUGAGUAGAGAACUGUGCUGGCGGGAGGGCCCCGGAACCGACCAGACGGAGGCAGAGGCACCCGCUGCCGUGCCCCCCCCUCAAGGAGGACCCCCUGGACCCUUUCUGGCCCACACCCACGCUGGUCUGCAGGCCCCAGGCCCACUGCCCGCCCCAGCGGGCGAUAAGGGUGACCUGCUCCUACAGGCGGUGCAACAGAGCUGUCUGGCCGACCACCUGUUGACCGCCAGCUGGGGGGCCGACCCGGUGCCCACCAAAGCUCCCGGAGAGGGCCAAGAAGGCCUCCCACUAACUGGCGCCUGCGCCGGGGGCCCGGGAUUACCCGCCGGCGAGCUGUAUGGCUGGGCCGUGGAGACCACGCCCAGCCCCGAGGGCGUGUCGUCCCUGGACAGCAGCAGCCUGAGCCUGUGCCUGAGCUCCGCCAACAGCCUGUAUGACGACAUCGAGUGCUUCCUGAUGGAGCUGGAACAACCCGCC(constitutively active truncated Hu IRF7 1-246 + 468-503; P032 withoutepitope tag) 218AUGGCACUGGCGCCUGAAAGAGCCGCUCCGCGUGUGCUCUUCGGCGAGUGGCUGCUGGGCGAGAUCAGCUCCGGCUGCUACGAGGGUCUACAGUGGCUGGACGAGGCCAGAACCUGUUUUAGAGUGCCCUGGAAGCACUUCGCGAGAAAGGACCUGAGCGAGGCCGACGCCAGAAUCUUCAAAGCCUGGGCAGUGGCUAGGGGCAGAUGGCCUCCCAGCAGCCGGGGCGGCGGCCCACCCCCCGAGGCCGAAACCGCCGAAAGAGCUGGCUGGAAGACCAACUUCAGAUGCGCCCUGAGAAGCACCAGAAGAUUUGUCAUGCUGAGAGAUAAUUCAGGAGACCCCGCCGACCCUCACAAGGUGUACGCCCUGUCCAGAGAGCUGUGUUGGAGAGAGGGCCCCGGAACCGACCAGACCGAGGCCGAGGCUCCAGCUGCCGUGCCACCCCCCCAAGGCGGACCACCCGGCCCCUUCUUGGCACAUACGCACGCCGGCCUCCAGGCUCCCGGCCCUCUGCCCGCCCCUGCUGGUGACAAAGGCGAUCUGCUGCUGCAAGCCGUCCAGCAAUCCUGCUUGGCUGACCACCUGCUGACCGCUAGCUGGGGAGCCGACCCCGUUCCCACCAAGGCUCCCGGAGAAGGACAGGAGGGCCUGCCCCUUACCGGCGCUUGCGCGGGGGGCCCUGGCUUGCCUGCCGGCGAACUGUACGGCUGGGCCGUGGAGACCACGCCUUCCCCCGAGGGCGUGUCCAGCCUGGACGAUGAUGACCUGGAUCUGUGCCUGGACAGCGCCAACGACCUGUACGAUGACAUCGAGUGCUUUUUGAUGGAGCUGGAGCAGCCCGCC(constitutively active truncated Hu IRF7 1-246 + 468-503 plusS475D/S476D/S477D/S479D/S483D/S487D; P036 without epitope tag) 219AUGGCCCUGGCCCCCGAGAGAGCCGCGCCCAGAGUGCUGUUCGGCGAAUGGCUGCUGGGCGAGAUCAGCAGCGGCUGCUAUGAGGGCCUGCAGUGGCUCGACGAAGCCAGGACGUGCUUCAGAGUCCCCUGGAAGCACUUCGCCAGAAAGGAUCUGAGCGAGGCUGACGCCAGAAUCUUCAAGGCCUGGGCAGUUGCGCGUGGGAGAUGGCCCCCCAGCUCGCGGGGCGGCGGUCCCCCCCCUGAGGCCGAGACCGCCGAAAGAGCCGGAUGGAAAACCAACUUUCGAUGCGCCCUCAGAAGCACCAGACGGUUUGUGAUGCUGAGAGAUAACAGCGGCGACCCUGCAGACCCCCAUAAAGUGUAUGCCCUGAGCAGAGAGCUGUGUUGGCGAGAGGGCCCCGGAACCGACCAAACCGAGGCCGAGGCCCCCGCCGCCGUACCCCCCCCUCAAGGCCCCCAGCCUGCUGCUCUGACCACGGGAGAAGCCGCCGCUCCUGAGAGCCCCCACCAAGCCGAGCCCUAUCUGAGCCCUAGCCCCAGCGCCUGCACCGCCGUGCAGGAGCCCUCACCGGGCGCCCUAGACGUGACCAUCAUGUACAAGGGGCGCACGGUGCUGCAAAAGGUGGUGGGCCACCCCAGCUGCACCUUCCUGUACGGCCCCCCCGACCCUGCCGUGAGAGCCACCGACCCCCAGCAAGUCGCCUUCCCCAGCCCCGCCGAGCUGCCCGACCAGAAGCAGCUGAGGUACACCGAGGAGUUGCUGAGACAUGUGGCCCCCGGCUUGCACCUCGAGCUGAGAGGCCCGCAGCUCUGGGCCAGAAGAAUGGGCAAGUGCAAGGUGUACUGGGAGGUGGGCGGCCCCCCCGGCAGCGCGAGCCCAAGCACCCCGGCCUGCCUGCUGCCUAGAAACUGCGACACCCCUAUCUUCGACUUCAGAGUAUUUUUCCAGGAGCUGGUCGAGUUCAGGGCCAGACAGCGUAGAGGCAGCCCCAGAUACACCAUCUACCUUGGAUUCGGCCAGGACCUGAGCGCCGGCAGACCCAAAGAGAAGUCCCUGGUACUGGUGAAGCUAGAGCCCUGGCUGUGUAGGGUGCAUCUGGAAGGCACCCAAAGAGAGGGCGUAAGCUCGCUUGACAGCAGCAGCCUCAGCCUGUGCCUGAGCAGCGCUAACAGCUUAUACGACGACAUCGAGUGCUUCCUGAUGGAGCUGGAACAACCCGCC (truncated Hu IRF7 1-151 + 247-503; P038 withoutepitope tag; null mutation) 220AUGGGCGGCCCUCCCGGGCCUUUCCUGGCCCAUACACACGCCGGCCUACAGGCUCCUGGCCCUCUGCCCGCCCCGGCCGGCGACAAGGGCGACCUCCUGCUGCAGGCCGUGCAGCAGUCCUGUCUGGCCGACCACCUGCUGACUGCUAGCUGGGGCGCCGAUCCCGUGCCCACCAAGGCCCCAGGAGAGGGGCAAGAGGGCCUGCCUCUAACCGGCGCAUGCGCAGGUGGACCAGGCCUCCCCGCCGGCGAGCUGUAUGGUUGGGCCGUGGAGACAACCCCCAGCCCCGGCCCGCAGCCUGCUGCGCUGACCACAGGCGAGGCCGCUGCCCCUGAGAGCCCCCACCAAGCUGAACCCUACCUGAGCCCCAGCCCCUCUGCCUGCACAGCGGUGCAGGAGCCCAGUCCCGGCGCCUUGGACGUGACCAUCAUGUAUAAGGGCAGGACUGUGUUACAAAAGGUAGUGGGCCACCCAAGUUGUACCUUUCUGUACGGGCCCCCCGACCCAGCCGUGCGCGCCACCGACCCCCAGCAGGUGGCCUUCCCCAGCCCCGCUGAGUUGCCCGAUCAGAAACAACUCCGGUACACCGAGGAAUUACUUAGACAUGUGGCUCCCGGCCUGCAUCUGGAGCUUAGAGGUCCACAGUUGUGGGCCAGAAGAAUGGGCAAGUGCAAGGUUUAUUGGGAGGUCGGAGGCCCCCCGGGCAGCGCCAGCCCCAGCACCCCCGCCUGUCUUCUGCCCAGAAACUGCGACACCCCAAUCUUCGAUUUCAGAGUGUUUUUCCAGGAACUGGUGGAGUUCAGAGCAAGGCAAAGAAGAGGCAGCCCUAGAUACACCAUCUACCUGGGCUUUGGCCAAGACCUGAGCGCCGGCAGACCCAAGGAAAAAUCCCUGGUCCUGGUGAAACUGGAGCCCUGGCUGUGCAGAGUCCACCUGGAGGGCACCCAGAGAGAGGGCGUGAGCAGCCUGGACUCGAGCAGCCUGUCCCUGUGUCUGAGCAGCGCGAAUUCGCUAUAUGACGACAUCGAAUGCUUUCUGAUGGAGCUGGAACAGCCCGCC(truncated Hu IRF7 152-503; P039 without epitope tag; null mutation) 221AUGCCUCACAGCAGCCUCCACCCUAGCAUCCCUUGCCCUAGAGGCCACGGCGCCCAGAAGGCCGCCCUCGUGCUUUUAAGCGCCUGCUUGGUGACCCUUUGGGGCUUGGGCGAGCCUCCAGAGCACACCUUGAGAUAUUUGGUGCUCCACCUGGCCAGCCUUCAGCUGGGCUUGUUACUCAACGGCGUGUGCAGCCUGGCCGAGGAGCUGAGACACAUCCACAGCAGAUACAGAGGCAGCUACUGGAGAACCGUGAGAGCGUGUCUGGGCUGCCCUCUGAGAAGAGGCGCCUUGCUUCUUCUCAGUAUCUACUUCUACUACUCCCUGCCUAACGCCGUGGGCCCUCCUUUCACCUGGAUGCUGGCACUGCUCGGCCUCAGCCAGGCCCUGAACAUCUUGUUGGGCUUGAAGGGCCUGGCCCCUGCCGAGAUCAGCGCCGUGUGCGAGAAGGGCAACUUCAACAUGGCCCACGGAUUGGCUUGGAGCUACUACAUCGGCUACCUGAGACUGAUCCUGCCUGAGCUGCAGGCCAGAAUCAGAACCUACAACCAGCACUACAACAACCUGCUGCGCGGCGCAGUGAGCCAGAGACUGUAUAUUCUGCUGCCUCUGGACUGCGGCGUGCCUGACAACCUGAGCAUGGCCGACCCUAACAUCAGAUUCCUGGACAAGCUGCCUCAGCAGACCGGCGACCACGCCGGCAUCAAGGACAGAGUGUACAGCAACAGCAUCUAUGAGCUGCUCGAGAAUGGCCAGAGAGCCGGCACCUGCGUGCUGGAGUACGCCACCCCUCUGCAGACCCUGUUCGCCAUGAGCCAGUAUAGUCAAGCUGGCUUCAGCAGAGAGGACAGACUGGAGCAGGCCAAGCUGUUCUGCAGAACCCUGGAGGACAUUCUGGCUGACGCCCCUGAGAGCCAGAACAACUGCCGACUGAUCGCCUACCAGGAACCAGCCGACGACAGCAGCUUCAGUCUUUCUCAGGAGGUUCUUCGCCACUUGCGCCAGGAGGAGAAGGAGGAGGUGACCGUGGGCAGCCUGAAGACCUCCGCAGUCCCUAGCACCAGCACCAUGAGUCAGGAGCCGGAGCUAUUAAUCAGCGGCAUGGAGAAGCCUCUUCCACUCCGAACCGACUUCAGCGCCACCAACUUCAGCCUGCUGAAGCAGGCAGGUGACGUUGAGGAGAAUCCGGGACCUAUGACCGAGUACAAGCUGGUGGUUGUGGGCGCCGACGGCGUGGGCAAGAGCGCCCUGACCAUCCAGCUGAUCCAG (KRAS(G12D)25mer_nt.STING(V155M)) 222AUGACCGAGUACAAGCUAGUAGUCGUGGGCGCCGACGGCGUGGGCAAGAGCGCCCUCACCAUCCAGCUAAUCCAGGCCACCAACUUCAGCUUGCUCAAGCAGGCCGGCGACGUGGAGGAGAACCCAGGCCCUAUGCCUCACAGCAGCCUUCACCCUAGCAUCCCUUGCCCUAGAGGCCACGGCGCCCAGAAGGCCGCCCUGGUGCUGCUGAGCGCCUGCCUGGUGACCCUGUGGGGCCUGGGCGAGCCUCCUGAGCACACCCUGAGAUAUCUGGUGCUUCACCUGGCCAGUUUACAGCUGGGCCUGCUUCUUAACGGCGUGUGCAGCCUGGCCGAGGAGCUGAGACACAUCCACAGCAGAUACAGAGGCAGCUACUGGAGAACCGUGAGAGCCUGCCUAGGCUGCCCUCUGAGAAGAGGCGCUCUGUUGCUACUUUCCAUCUACUUCUACUACUCCCUGCCUAACGCCGUGGGCCCUCCUUUCACUUGGAUGCUGGCGUUGCUGGGUCUGAGCCAGGCCCUGAACAUCCUUCUCGGUCUGAAGGGCCUGGCCCCUGCCGAGAUCAGCGCCGUGUGCGAGAAGGGCAACUUCAACAUGGCCCACGGACUCGCCUGGAGCUACUACAUCGGCUACCUGAGACUGAUCCUGCCUGAGCUGCAGGCCAGAAUCAGAACCUACAACCAGCACUACAACAACCUGCUGCGGGGCGCCGUGAGCCAGAGACUGUAUAUACUUCUUCCUCUGGACUGCGGCGUGCCUGACAACCUGAGCAUGGCCGACCCUAACAUCAGAUUCCUGGACAAGCUGCCUCAGCAGACCGGCGACCACGCCGGCAUCAAGGACAGAGUGUACAGCAACUCCAUUUAUGAGCUGCUCGAGAAUGGCCAGAGAGCCGGCACCUGCGUGCUGGAGUACGCCACCCCUCUGCAGACCCUGUUCGCCAUGAGCCAGUACAGUCAGGCUGGAUUCAGCAGAGAGGACAGACUGGAGCAGGCCAAGCUGUUCUGCAGGACACUGGAGGACAUACUAGCAGACGCCCCUGAGAGCCAGAACAACUGCAGACUGAUUGCCUACCAGGAGCCUGCGGACGACAGCUCCUUCAGUCUGAGUCAGGAGGUGUUGCGGCACUUACGCCAAGAAGAGAAGGAGGAGGUGACCGUGGGCAGCCUGAAGACUAGCGCUGUGCCUAGCACCAGCACAAUGUCACAGGAGCCGGAAUUGCUAAUCAGCGGCAUGGAGAAGCCUCUCCCAUUACGUACCGACUUCAGC (KRAS(G12D)25mer_ct.STING(V155M)) 223AUGCCUCACAGCAGCCUUCACCCUAGCAUCCCUUGCCCUAGAGGCCACGGCGCCCAGAAGGCCGCCCUAGUGCUCCUUAGCGCCUGCCUCGUGACCCUAUGGGGCUUAGGCGAGCCUCCAGAGCACACCUUGAGAUACCUCGUCCUCCACCUGGCUAGUCUACAGCUGGGCCUUCUCCUCAACGGCGUGUGCAGCCUGGCCGAGGAGCUGAGACACAUCCACAGCAGAUACAGAGGCAGCUACUGGAGAACCGUGAGAGCGUGCCUGGGCUGCCCUCUGAGAAGAGGCGCACUGCUGUUACUCAGCAUCUACUUCUACUACUCACUGCCAAACGCCGUGGGCCCUCCUUUCACCUGGAUGCUGGCCUUGCUCGGAUUGAGCCAGGCCCUGAACAUUUUACUGGGAUUGAAGGGCCUGGCCCCUGCCGAGAUCAGCGCCGUGUGCGAGAAGGGCAACUUCAACAUGGCCCACGGCCUAGCUUGGAGCUACUACAUCGGCUACCUGAGACUGAUCCUGCCUGAGCUGCAGGCCAGAAUCAGAACCUACAACCAGCACUACAACAACCUGCUGCGUGGAGCGGUGAGCCAGAGACUGUAUAUCCUCCUGCCUCUGGACUGCGGAGUGCCUGACAACCUGAGCAUGGCCGACCCUAACAUCAGAUUCCUGGACAAGCUGCCUCAGCAGACCGGCGACCACGCCGGCAUCAAGGACAGAGUGUACAGCAACUCAAUCUACGAGCUGUUGGAGAAUGGCCAGAGAGCCGGCACCUGCGUGCUGGAGUACGCCACCCCUCUGCAGACCCUGUUCGCCAUGAGCCAGUACUCUCAGGCAGGCUUCAGCAGAGAGGACAGACUGGAGCAGGCCAAGCUGUUCUGCAGAACCCUGGAGGACAUCCUGGCGGACGCCCCUGAGAGCCAGAACAACUGCCGGCUUAUCGCCUACCAGGAGCCAGCAGACGACAGCAGCUUCUCUCUCUCACAAGAGGUACUGCGCCAUCUUCGCCAGGAGGAGAAGGAGGAGGUGACCGUGGGCAGCCUGAAGACAUCCGCCGUACCUAGCACCAGCACCAUGUCUCAGGAACCGGAACUGUUGAUCAGCGGCAUGGAGAAGCCUCUGCCACUGCGCACCGACUUCAGCGCCACCAACUUCUCCCUACUGAAGCAAGCCGGUGACGUUGAAGAGAACCCUGGCCCUAUGACCGAGUACAAGCUGGUAGUAGUAGGCGCCGACGGCGUGGGCAAGAGCGCCCUGACCAUCCAGCUGAUCCAGAUGACUGAAUAUAAGCUUGUCGUCGUGGGCGCAGAUGGCGUUGGUAAGAGCGCACUUACAAUUCAACUCAUUCAGAUGACGGAGUAUAAGCUGGUGGUGGUCGGAGCUGACGGCGUAGGCAAGAGUGCCCUUACUAUUCAGCUAAUUCAG (KRAS(G12D)25mer^3_nt.STING(V155M)) 224AUGACCGAGUACAAGCUUGUGGUGGUUGGCGCCGACGGCGUGGGCAAGAGCGCCUUAACCAUCCAGCUUAUCCAGAUGACAGAGUAUAAGCUAGUGGUGGUCGGCGCAGACGGAGUGGGAAAGAGUGCAUUAACUAUUCAACUCAUCCAAAUGACCGAAUACAAGCUAGUAGUUGUGGGUGCAGAUGGCGUCGGCAAGUCUGCACUGACAAUUCAGCUCAUCCAGGCCACCAACUUCAGCCUGCUGAAGCAGGCCGGCGACGUGGAGGAGAACCCUGGCCCUAUGCCUCACAGCAGCCUGCACCCUAGCAUCCCUUGCCCUAGAGGCCACGGCGCCCAGAAGGCCGCCCUGGUGCUGCUGAGCGCCUGCCUGGUGACCCUGUGGGGCCUGGGCGAGCCUCCUGAGCACACCCUGAGAUACCUAGUUUUGCACCUGGCUUCUCUGCAGCUGGGCCUACUGCUCAACGGCGUGUGCAGCCUGGCCGAGGAGCUGAGACACAUCCACAGCAGAUACAGAGGCAGCUACUGGAGAACCGUGAGAGCAUGCUUAGGCUGCCCUCUGAGAAGAGGCGCUCUGCUCCUCUUGUCCAUCUACUUCUACUACUCGCUACCUAACGCCGUGGGCCCUCCUUUCACCUGGAUGCUGGCCCUCUUGGGAUUAAGCCAGGCCCUGAACAUCUUGCUGGGACUGAAGGGCCUGGCCCCUGCCGAGAUCAGCGCCGUGUGCGAGAAGGGCAACUUCAACAUGGCCCACGGACUCGCUUGGAGCUACUACAUCGGCUACCUGAGACUGAUCCUGCCUGAGCUGCAGGCCAGAAUCAGAACCUACAACCAGCACUACAACAACCUGCUGCGGGGAGCAGUGAGCCAGAGACUGUAUAUUCUGCUCCCUCUGGACUGCGGCGUGCCUGACAACCUGAGCAUGGCCGACCCUAACAUCAGAUUCCUGGACAAGCUGCCUCAGCAGACCGGCGACCACGCCGGCAUCAAGGACAGAGUGUACAGCAACAGCAUUUACGAGCUGCUGGAGAACGGCCAGAGAGCCGGCACCUGCGUGCUGGAGUACGCCACCCCUCUGCAGACCCUGUUCGCCAUGAGCCAGUACUCCCAGGCAGGAUUCAGCAGAGAGGACAGACUGGAGCAGGCCAAGCUGUUCUGCCGUACUCUUGAGGACAUCCUUGCAGACGCCCCUGAGAGCCAGAACAACUGCCGGUUGAUUGCCUACCAGGAACCGGCAGACGACAGCUCAUUCUCCUUGUCUCAGGAGGUCCUUAGACACCUGCGGCAGGAGGAGAAGGAGGAGGUGACCGUGGGCAGCCUGAAGACAUCCGCCGUGCCUAGCACGUCUACCAUGUCCCAGGAGCCGGAACUGCUAAUCAGCGGCAUGGAGAAGCCUCUGCCUCUCAGGACCGACUUCAGC (KRAS(G12D)25mer^3_ct.STING(V155M)) 225AUGCCCCAUAGCAGCCUGCACCCCAGCAUCCCCUGCCCCAGAGGCCACGGCGCCCAGAAGGCCGCCCUGGUCCUGCUGAGCGCAUGCCUGGUCACCCUGUGGGGCCUGGGCGAGCCCCCCGAGCACACCCUGAGAUACCUGGUGCUGCACCUCGCCAGCCUGCAGCUGGGCCUGCUGCUGAACGGCGUGUGCAGCCUGGCCGAGGAGCUGAGACACAUCCACAGCAGAUAUAGAGGCAGCUACUGGAGAACCGUGAGAGCUUGCCUCGGCUGCCCCCUGAGAAGAGGCGCCCUGCUGCUGCUGAGCAUCUACUUUUACUACAGCCUGCCCAACGCUGUGGGCCCCCCUUUCACGUGGAUGCUCGCCCUGCUGGGACUGAGCCAGGCCCUGAACAUCCUGCUGGGCCUUAAGGGCCUAGCCCCCGCCGAGAUCAGCGCCGUGUGCGAGAAGGGCAACUUCAAUGUGGCCCACGGCCUGGCCUGGAGCUACUACAUCGGCUACCUGAGACUGAUCCUGCCCGAGCUGCAGGCCAGAAUCAGAACCUACAAUCAGCACUACAACAACCUGCUGAGAGGCGCCGUGAGCCAGAGACUGUACAUCCUGCUGCCCCUGGACUGCGGCGUGCCCGACAACCUCAGCAUGGCCGACCCCAACAUCAGAUUCCUGGACAAGCUGCCCCAGCAGACCGGCGACCACGCCGGCAUCAAGGAUCGCGUGUACAGCAACAGCAUCUACGAGCUGCUGGAAAACGGCCAGAGAGCCGGAACCUGCGUGCUGGAGUACGCCACACCCCUGCAGACCCUGUUCGCCAUGAGCCAGUACAGCCAGGCCGGCUUCAGCAGAGAGGACAAGCUGGAGCAGGCCAAGCUGUUCUGCAGAACCCUGGAGGAUAUCCUCGCCGACGCCCCCGAGAGCCAGAACAACUGCAGGCUGAUCGCGUACCAGGAGCCCGCUGACGACAGCAGCUUUAGCCUGAGCCAGGAGGUGCUGAGACAUCUGCGUCAAGAGGAAAAGGAGGAGGUGACCGUGGGCUCCCUGAAGACCAGCGCCGUGCCCAGCACCAGCACCAUGAGCCAGGAGCCCGAGCUGCUGAUCAGCGGCAUGGAGAAGCCACUGCCCCUCAGAACCGACUUCAGCACC (Hu STING (R284K) var; no epitope tag) 226 ATIGTAMYK (EBV BRLF1peptide) 227 SIIPSGPLK (FLU peptide) 228 AVDLSHFLK (HIV NEF peptide) 229AVFDRKSDAK (EBV peptide) 230 YVNVNMGLK (HBV core antigen peptide) 231RVCEKMALY (HC peptide) 232 KLGGALQAK (CMV peptide)

1. An immunomodulatory therapeutic composition, comprising: an mRNAcomprising an open reading frame encoding a concatemer of two or moreactivating oncogene mutation peptides.
 2. (canceled)
 3. Theimmunomodulatory therapeutic composition of claim 1, wherein at leastone of the activating oncogene mutation peptides is a KRAS mutationpeptide, optionally wherein the KRAS mutation peptide comprises a G12mutation and/or a G13 mutation, optionally wherein the G12 KRAS mutationis selected from (i) G12D, G12V, G12S, G12C, G12A, and G12R KRASmutations; or (ii) G12D, G12V, and G12C KRAS mutations, optionallywherein the G13 KRAS mutation is a G13D KRAS mutation. 4-10. (canceled)11. The immunomodulatory therapeutic composition of claim 3, wherein theconcatemer comprises 3, 4, 5, 6, 7, 8, 9, or 10 activating oncogenemutation peptides, optionally wherein the concatemer comprises 4activating oncogene mutation peptides.
 12. (canceled)
 13. Theimmunomodulatory therapeutic composition of claim 11, wherein theconcatemer comprises activating oncogene mutation peptides comprising,individually, G12D, G12V, G12C, and G13D KRAS mutations. 14-16.(canceled)
 17. The immunomodulatory therapeutic composition of claim 3,wherein the activating oncogene mutation peptides comprise,individually, 10-30, 15-25, or 20-25 amino acids in length, or whereinthe activating oncogene mutation peptides comprise, individually, 20,21, 22, 23, 24, or 25 amino acids in length. 18-53. (canceled)
 54. Theimmunomodulatory therapeutic composition of claim 3, wherein theconcatemer comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 42-47, 73 and 137, or wherein the mRNAencoding the concatemer comprises a nucleotide sequence selected fromthe group consisting of SEQ ID NOs: 129-131, 133, 138 and
 169. 55.(canceled)
 56. The immunomodulatory therapeutic composition of claim 54,wherein the mRNA includes at least one chemical modification, optionallywherein the chemical modification is selected from the group consistingof pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4′-thiouridine,5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine,2-thio-dihydropseudouridine, 2-thio-dihydrouridine,2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine,4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine,4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine,5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2′-O-methyluridine, or wherein the chemical modification is selected from the groupconsisting of pseudouridine or a pseudouridine analog, or wherein thechemical modification is N1-methylpseudouridine. 57-69. (canceled)
 70. Alipid nanoparticle comprising the immunomodulatory therapeuticcomposition of claim 1, optionally wherein the lipid nanoparticlecomprises a molar ratio of about 20-60% ionizable amino lipid:5-25%phospholipid:25-55% sterol, and 0.5-15% PEG-modified lipid, optionallywherein the ionizable amino lipid is a compound of Formula (I),optionally wherein the compound of Formula (I) is Compound
 25. 71-106.(canceled)
 107. The lipid nanoparticle of claim 70, wherein the lipidnanoparticle comprises a molar ratio of about 50% Compound 25:about 10%DSPC:about 38.5% cholesterol; and about 1.5% PEG-DMG. 108-119.(canceled)
 120. A pharmaceutical composition comprising theimmunomodulatory therapeutic composition of claim 1, and apharmaceutically acceptable carrier, optionally wherein thepharmaceutically acceptable carrier comprises a buffer solution.
 121. Apharmaceutical composition comprising the lipid nanoparticle of claim70, and a pharmaceutically acceptable carrier, optionally, wherein thepharmaceutically acceptable carrier comprises a buffer solution. 122.The pharmaceutical composition of claim 120, which is formulated forintramuscular delivery. 123-124. (canceled)
 125. A kit comprising acontainer comprising the immunomodulatory therapeutic composition ofclaim 1, and an optional pharmaceutically acceptable carrier, and apackage insert comprising instructions for administration of theimmunomodulatory therapeutic composition, the lipid nanoparticle orpharmaceutical composition, for treating or delaying progression ofcancer in an individual.
 126. The kit of claim 125, wherein the packageinsert further comprises instructions for administration of theimmunomodulatory therapeutic composition in combination with acomposition comprising a checkpoint inhibitor polypeptide, and anoptional pharmaceutically acceptable carrier, for treating or delayingprogression of cancer in an individual. 127-134. (canceled)
 135. Amethod of reducing or decreasing a size of a tumor, inhibiting a tumorgrowth, or inducing an anti-tumor response in a subject in need thereof,comprising administering to the subject the immunomodulatory therapeuticcomposition of claim
 1. 136. The method of claim 135, wherein theimmunomodulatory therapeutic composition is administered in combinationwith a cancer therapeutic agent, or wherein the immunomodulatorytherapeutic composition is administered in combination with aninhibitory checkpoint polypeptide or polynucleotide encoding the same,optionally wherein the inhibitory checkpoint polypeptide is an antibodyor an antigen-binding fragment thereof that specifically binds to amolecule selected from the group consisting of PD-1, PD-L1, TIM-3,VISTA, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR and LAG3. 137-138.(canceled)
 139. The method of claim 135, wherein the cancer is selectedfrom a cancer of the pancreas, peritoneum, large intestine, smallintestine, biliary tract, lung, endometrium, ovary, genital tract,gastrointestinal tract, cervix, stomach, urinary tract, colon, rectum,and hematopoietic and lymphoid tissues. 140-189. (canceled)
 190. Thepharmaceutical composition of claim 121, which is formulated forintramuscular delivery.
 191. The immunomodulatory therapeuticcomposition of claim 1, wherein the concatemer comprises the amino acidsequence of SEQ ID NO:
 137. 192. The immunomodulatory therapeuticcomposition of claim 1, wherein the ORF comprises the nucleotidesequence of SEQ ID NO:
 169. 193. The immunomodulatory therapeuticcomposition of claim 1, wherein the mRNA comprises the nucleotidesequence of SEQ ID NO:
 167. 194. The immunomodulatory therapeuticcomposition of claim 193, wherein the mRNA comprises at least onechemical modification that is N1-methylpseudouridine.